JP6250556B2 - Pyrimido [4,5-b] indole derivatives and their use in the proliferation of hematopoietic stem cells - Google Patents

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Cross-reference of related applications
[0001] This application claims the benefit of priority under US Provisional Application No. 61 / 591,521, filed Jan. 27, 2012. The entire disclosure of this application is incorporated herein by reference in its entirety.

  [0002] The present invention relates to pyrimido [4,5-b] indole derivatives. The invention also relates to the use of pyrimido [4,5-b] indole derivatives for the growth of hematopoietic stem cells. The present invention further relates to medical treatment of diseases involving hematopoietic stem cells.

  [0003] The main sources of hematopoietic stem cells (HSC) are bone marrow and umbilical cord blood (UCB). HSCs cure in patients with malignant blood diseases, bone marrow failure states, various congenital diseases of global concern (eg sickle cell anemia and thalassemia (Mediterranean anemia)), and autoimmune diseases such as lupus Used in the context of transplantation (autologous or allogeneic transplantation) that constitutes one of the most effective treatment strategies to achieve this. However, this opportunity for lifesaving or life improvement treatment is available to thousands of people around the world because these cells cannot be amplified ex vivo enough to safely and successfully perform the surgery. It has not been. More specifically, one in every three patients has forgotten the transplant opportunity because no human leukocyte antigen (HLA) compatible donor is found. Another percentage of patients do not have a transplant opportunity simply because too little HSC is available in the graft (ie, cord blood or autograft) and a successful transplant cannot be expected. The safety and effectiveness of bone marrow transplantation is directly dependent on the number of HSCs and progenitor cells available for engraftment. The more infused, the faster blood function is restored and the risk of infection due to granulocyte deficiency or the bleeding time frame due to platelet deficiency is shorter. The challenge of providing sufficient HSC is that a non-myeloablative pretreatment is suitable, such as the situation of gene therapy for major hereditary blood diseases (a major genetic cause of morbidity and mortality worldwide) The case is even higher.

  [0004] In adults, because HSCs are primarily present in the bone marrow, both autologous and allogeneic hematopoietic stem cell transplantation (HSCT) must first be mobilized into the circulation before harvesting by apheresis. Since the amount of CD34 + cells, a surrogate marker for (HSC), affects the success and rate of hematopoietic recovery, it is most important to collect an appropriate number of CD34 + cells. Several reports suggest that the greater the injected dose of CD34 + cells, the better the improvement in survival is predicted.

  [0005] The two most commonly used mobilization methods are granulocyte colony stimulating factor (G-CSF) and G-CSF + chemotherapy. The CXCR4 antagonist Plerixafor, approved by the US Food and Drug Administration (FDA) in 2008 and in 2011 by Health Canada, mobilizes HSC when administered with G-CSF. To strengthen. However, prelixafor is a contraindication for leukemia patients because it mobilizes leukemia cells. It is estimated that up to 15% of patients are affected by the currently used mobilization method failing to obtain a sufficient number of CD34 + cells / kg (variations between diseases). The use of autologous HSCT in malignant blood diseases is often limited by the fact that both normal and cancer stem cells are present in the bone marrow and possibly mobilized.

  [0006] Allogeneic HSCT with BM (bone marrow) or mPBSC (mobilized peripheral blood stem cells) is another transplant option. However, about a third to a quarter of patients suitable for this type of transplant cannot find a suitable donor. In those who have undergone transplantation, there is a high frequency of transplant-related deaths due to the risk of graft-versus-host disease, relapse or transplant rejection; and long-term immunodeficiency. Alternatively, umbilical cord blood has been shown as an effective option in allogeneic HSCT. However, a single CB unit usually provides insufficient HSC for adult patients for rapid and efficient hematopoietic recovery.

  [0007] In vitro conditions that support cytokine-mediated short-term maintenance or even mild increases in the number of mouse or human HSCs as measured by mouse reconstitution assays generally have a much stronger increase in late-type progenitor cell populations. accompanied by increase). A more prominent increase in mouse and human HSCs has recently been in cultures containing other factors such as fibroblast growth factor (FGF), insulin-like growth factor binding protein, angiopoietin-like growth factor and pleiotrophin. It is reported in things. However, these latter reports have been solitary so far and are awaiting third party confirmation. Short-term increases in HSCs obtained with standard cytokines in vitro also inevitably lead to depletion of final HSCs.

  [0008] Alternatives for human HSC growth include culturing them with stromal elements or soluble morphogenic ligands (eg stimulation of Notch, Wnt and Hedgehog pathways), specific Targeted manipulation of intracellular signaling pathways (PGE2, ROS, p38 and MAPK inhibitors) or manipulation of specific transcription factors (eg, Hox, Hlf) has been required. Other preclinical means for ex vivo growth of HSCs are: i) StemRegenin1 (SR1, aryl hydrocarbon receptor antagonist) (Boitano, AE et al., “Aryl hydrocarbon receptor antagonist promotes proliferation of human hematopoietic stem cells (Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells) ”, Science 329: 1345-1348.2010); ii) Garcinol (histone acetyltransferase inhibitor) (Nishino, T et al.,“ a potent inhibitor of histone acetyltransferase. ” Ex vivo expansion of human hematopoietic stem cells by Garcinol, Garcinol, a potent inhibitor of histone acetyltransferase "PLoS ONE 6 (9): e24298.2011 And iii) NR-101 (non-peptidyl small molecule c-MPL agonist) (Nishino et al., “Ex vivo expansion of human hematopoietic stem cells by a small- molecule agonist of c-MPL) "Exp.Hem.2009; 37: 1364-1377). The characterization of SR1 proved the principle that low molecular weight (LMW) compounds have the ability to promote HSC growth.

[0009] Clinical studies emphasize not only the persistence of administered transplants but also the requirement for the importance of minimizing the time to appearance of useful granulocyte levels after transplantation. This is dependent on the number of short-term repopulated cells injected. Transplantation of bone marrow or umbilical cord blood cells grown in culture with cytokines so far has not shown a clinically useful acceleration of hematopoietic recovery compared to untreated cells. Initial test results using cells grown with immobilized Notch ligands show for the first time the potential clinical utility of any (if any) progenitor cell proliferation strategy (Delaney et al., “Rapid” Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution ", Nat. Med. 16 (2): 232-236.2010 ). However, this approach is limited by the need to use immobilized delta-1 fusion protein during the ex vivo growth process and the lack of documented (demonstrated) effects on stem cells (effects are more differentiated) Seems to be limited to progenitor cells). Other efforts in clinical trials are as follows. i) Combination of StemEx, copper chelator tetraethylenepentamine (TEPA) and cytokines and cultured UCB cells, co-injection with untreated UCB cells; Phase I results up to neutrophil or platelet engraftment (De Lima M et al., “Transplantation of cord blood cells ex vivo grown with the copper chelator tetraethylenepentamine; phase I / II study). (Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I / II clinical trial) ”, Bone Marrow Transplant. 2008; 41 (9): 771-778); and 16-16 dimethylprostaglandin E2 (PGE2), used to improve homing of UCBT in Phase I trials.

  [0010] Thus, there is a need for new strategies to increase the proliferation of hematopoietic stem and progenitor cells. Certain pyrimido [4,5-b] indole derivatives used in that context are known in the art. They are for example WO 2003/037898; WO 2004/058764; WO 1998/042708; WO 1997/002266; WO 2000/066655; WO 1993/020078; WO 2006/116733; WO 2008/055233; WO 2010/006032; 1995/019970; WO 2005/037825; and WO 2009/004329. However, these references do not disclose the pyrimido [4,5-b] indole derivatives according to the present invention nor their use in the proliferation of hematopoietic stem and progenitor cells.

International Patent Publication No. WO 2003/037898 International Patent Publication No. WO 2004/058764 International Patent Publication No. WO 1998/042708 International Patent Publication No. WO 1997/002266 International Patent Publication No. WO 2000/066585 International Patent Publication No. WO 1993/020078 International Patent Publication No. WO 2006/116733 International Patent Publication No. WO 2008/055233 International Patent Publication No. WO 2010/006032 International Patent Publication No. WO 1995/019970 International Patent Publication No. WO 2005/037825 International Patent Publication No. WO 2009/004329

Boitano, AE et al., Science 329: 1345-1348.2010 Nishino, T et al., PLoS ONE 6 (9): e24298.2011 Nishino et al., Exp. Hem. 2009; 37: 1364-1377 Delaney et al., Nat. Med. 16 (2): 232-236.2010 de Lima M et al., Bone Marrow Transplant. 2008; 41 (9): 771-778

  [0019] The inventors have discovered certain pyrimido [4,5-b] indole derivatives. These compounds are useful for the growth of hematopoietic stem cell populations, particularly human hematopoietic stem cell populations. The compound is also useful for medical treatment of diseases involving hematopoietic stem cells.

  [0020] According to one aspect, the present invention provides compounds of the following general formulas I, II, III, IV, V and VI.

[0021] Substituents in the above general formulas I, II, III, IV, V and VI, namely Z, W, L, Li, X, X _i , R ¹ , R ² , R ³ , R ⁴ and m is as defined below.
[0022] According to one aspect, the present invention provides a pharmaceutical composition comprising a compound of general formula I, II, III, IV, V or VI.

  [0023] According to one aspect, the present invention provides the use of a compound of general formula I, II, III, IV, V or VI for growing hematopoietic stem cells. In an embodiment of the invention, the hematopoietic stem cell is a human cell.

  [0024] According to one aspect, the present invention provides a method of increasing hematopoietic stem cells or progenitor cells. The method comprises culturing the starting cell population in the presence of a compound of general formula I, II, III, IV, V or VI. In embodiments of the invention, the starting cell population is in vivo, in vitro or ex vivo. Also, in an embodiment of the invention, the starting cell population comprises CD34 + cells taken from mobilized peripheral blood (mPB), bone marrow (BM) or umbilical cord blood (UCB). Furthermore, in an embodiment of the method according to the invention, the cultivation of the starting cell population in the presence of a compound of general formula I, II, III, IV, V or VI is at least one cell which is a biomolecule or another small molecule. It may be performed with growth factors.

  [0025] According to one aspect, the present invention provides a cell population expanded according to the method of the present invention, more particularly a cell population expanded using a compound according to the present invention. In an embodiment, the present invention provides hematopoietic stem cells grown according to the method of the present invention, and more particularly hematopoietic stem cells grown using a compound according to the present invention.

  [0026] According to one aspect, the present invention provides a method for treating a hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immune deficiency disease in a subject. The method comprises a hematopoietic stem cell expanded with a compound of general formula I, II, III, IV, V or VI, or a general formula I, II, III, IV, Administration of a compound of V or VI.

  [0027] In an embodiment of the invention, the hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immunodeficiency disease is a bone marrow failure condition, various congenital diseases of global interest (eg sickle-like). Red blood cell anemia and thalassemia), lupus, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myeloproliferative disorder, myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Includes Hodgkin's disease, aplastic anemia, atypical erythropoiesis, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiscott-Aldrich syndrome, congenital metabolic disorders (particularly Gaucher's disease).

  [0028] According to one aspect, the present invention provides a kit for use in increasing stem or progenitor cells, or for growing hematopoietic stem cells. The kit includes a compound of general formula I, II, III, IV, V or VI and instructions for use. In an embodiment of the invention, the kit comprises at least one cell growth factor that is a biomolecule or another small molecule.

[0011] FIG. 1 shows that Compound 1 is not acting through an aryl hydrocarbon (AhR) pathway. Mobilized peripheral blood CD34 (+) cells were cultured with DMSO, SR1 [AhR antagonist 1000 nM], and Compound 1 [500 nM] for 12 hours, cells were harvested and real-time quantitative for AhR-responsive genes (CYP1B1 and AhRR) RT-PCR was performed. Compound 1, unlike SR1, does not repress the AhR-downstream target gene, suggesting that its function is independent of the AhR pathway. [0012] FIG. 2 shows that the action of Compound 1 is reversible. Mobilized peripheral blood CD34 (+) cells cultured with Compound 1 (shown in green) or vehicle (DMSO, shown in blue) for 7 days and washed with or without compound in fresh medium ( (Solid line vs. dotted line) Cells were cultured for an additional 8 days and the percentage of CD34 + CD45RA− was monitored. When Compound 1 was washed off, a sharp decrease in the percentage of CD34 + CD45RA- was observed, indicating that the effect was reversible. [0013] FIG. 3 shows that Flt3, SCF, and TPO are required for compound 1-mediated stem cell proliferation. Mobilized peripheral blood CD34 (+) cells were cultured for 7 days in the presence or absence of growth factors (Flt3 + SCF + TPO). CD34 + CD45RA− cell numbers are low in the absence of any growth factors, suggesting that they are required for the observed effects. [0014] FIG. 4A shows that Compound 1 reduces differentiation of CD34 + mobilized peripheral blood cells. As determined by FACS analysis, Compound 1 was able to expand the CD34 + cell population compared to DMSO treated control cells. SR1 synergizes with Compound 1 to maintain proliferating cultured cell CD34 +, suggesting that inhibition of differentiation is even more pronounced with the combination. FIG. 4B shows that Compound 1 reduces CD34 + cord blood cell differentiation. FIG. 4C shows that compound 40 reduces CD34 + cord blood cell differentiation. FIG. 4D shows that compound 40 has a dose-dependent effect on the inhibition of CD34 + cell differentiation. [0015] FIG. 5A shows that Compound 1 expands mobilized peripheral blood-derived CD34 + and CD34 + CD45RA− cell populations ex vivo. Total cell numbers were the same in DMSO and compound 1 treated cells, but the CD34 + population was more than twice that in DMSO. Similarly, the CD34 + CD45RA− population was more than 3-fold in the Compound 1 treatment group versus DMSO. This observation was enhanced by co-treatment with SR1. FIG. 5B shows that Compound 1 enhances the proliferation of cord blood-derived CD34 + and CD34 + CD45RA− cells during 7 days of culture. FIG. 5C shows that Compound 1 exerts a positive effect on in vitro growth of cord blood-derived CD34 + and CD34 + CD45RA− cells during 12 days of culture. [0016] FIG. 6 shows that CD34 + mPB cells were cultured for 10 days in the presence of vehicle (DMSO), SR1, Compound 1, and a combination of Compound 1 and SR1. Results from 50,000 and 500,000 treated cells were transplanted into NSG mice and bone marrow analysis was performed 13 weeks after transplantation to assess human hematopoietic cell engraftment. CD34 + mPB cells treated with Compound 1 took better than DMSO at any given cell dose. Interestingly, the percentage of human CD45 + cells in the bone marrow was highest in NSG mice transplanted with cells treated with the combination (Compound 1 + SR1) compared to the individual compounds. [0017] FIG. 7A shows that Compound 1 proliferating cells can reconstitute human hematopoietic cells in NSG mice. Outcomes of 5,000 CD34 + CB cells treated with vehicle (DMSO), SR1, compound 1 and combinations were transplanted into immunodeficient NSG mice. Eight weeks after transplantation, bone marrow analysis showed human CD45 + engraftment in all treatment groups except DMSO. Cells treated with the combination (Compound 1 + SR1) showed the highest engraftment level. FIG. 7B shows that, during 12 days in vitro culture, compound 40 prevents the loss of human hematopoietic cells that have the ability to engraft in the bone marrow of NSG mice. [0018] FIG. 8 shows the proliferation of undifferentiated cell phenotypes (CD34 ⁺ , CD34 ⁺ CD90 ⁺ , and CD34 ⁺ CD45RA ⁺ ) in a bioreactor using a fed-batch mode, as measured by flow cytometry. FB control = fed-batch culture without compound 40, Cpd 40 = fed-batch culture with compound 40.

  [0029] The inventors have discovered certain pyrimido [4,5-b] indole derivatives. These compounds are useful for the growth of hematopoietic stem cell populations, particularly human hematopoietic stem cell populations. The compound is also useful for medical treatment of diseases involving hematopoietic stem cells.

  [0030] The compounds according to the invention have the general formulas I, II, III, IV, V and VI shown below. Salts or prodrugs of such compounds are also within the scope of the compounds according to the invention.

[0031] In formulas I, II, III, IV, V and VI, the substituents are defined as outlined below.
[0032] Z is: 1) -P (O) (OR ¹ ) (OR ¹ ), 2) -C (O) OR ¹ , 3) -C (O) NHR ¹ , 4) -C (O) N (R ¹ ) R ¹ , 5) -C (O) R ¹ , 6) -CN, 7) -SR ¹ , 8) -S (O) ₂ NH ₂ , 9) -S (O) ₂ NHR ¹ , _{^{10) -S (O) 2 N}} (R 1) R 1, 11) -S (O) R 1, 12) -S (O) 2 R 1, 13) -L, 14) - benzyl (1,2 Or optionally substituted with three R ^A or R ¹ substituents), 15) -L-heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents, substituted The group is bound to either or both of the L and heteroaryl groups), 16) -L-heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents; Group And are coupled to one or both of the heterocyclyl group), 17)-L-aryl (it may be substituted by one or more R ^A or R ¹ substituent, the substituents L and is coupled to either or both of the aryl group), 18) - may be substituted with a heteroaryl (one or more R ^a or R ¹ substituent), or 19) - aryl (1 Or optionally substituted with one or more R ^A or R ¹ substituents. In this list, each substituent may be attached to the L group if it is not already present; when (R ¹ ) and R ¹ are attached to a nitrogen atom, they are taken together with the nitrogen atom. To form a 3- to 7-membered ring which may contain one or more other heteroatoms selected from N, O and S, and the ring contains one or more rings. It may be substituted with R ¹ or R ^A.

[0033] W is a H, halogen, or group attached to the pyrimidoindole nucleus of the molecule through an N, O, S, or C atom. Optionally, W may comprise at least one moiety that is saturated, unsaturated, linear, branched and / or cyclic alkyl and / or heteroalkyl having 1 to 20 carbon atoms. Also optionally, the moiety may contain at least one other heteroatom that is N, O or S. As will be appreciated by those skilled in the art, W in the chemical structure of the compounds according to the invention may belong to various categories of chemical groups commonly used in the art.

[0034] More particularly, W is: 1) -H, 2) - ^{halogen, 3) -OR 1, 4)} -L-OH, 5) -L-OR 1, 6) -SR 1, 7) - ^{CN, 8) -P (O)} (OR 1) (OR 1), 9) -NHR 1, 10) -N (R 1) R 1, 11) -L-NH 2, 12) -L-NHR 1 13) -LN (R ¹ ) R ¹ , 14) -L-SR ¹ , 15) -LS (O) R ¹ , 16) -LS (O) ₂ R ¹ , 17)- ^{L-P (O) (OR} 1) (OR 1), 18) -C (O) OR 1, 19) -C (O) NH 2, 20) -C (O) NHR 1, 21) -C ( O) N (R ¹ ) R ¹ , 22) -NHC (O) R ¹ , 23) -NR ¹ C (O) R ¹ , 24) -NHC (O) OR ¹ , 25) -NR ¹ C (O ^{) OR 1, 26) -OC (} O) NH ₂ , 27) -OC (O) NHR ¹ , 28) -OC (O) N (R ¹ ) R ¹ , 29) -OC (O) R ¹ , 30) -C (O) R ¹ , 31)- ^{_{NHC (O) NH 2, 32}} ) -NHC (O) NHR 1, 33) -NHC (O) N (R 1) R 1, 34) -NR 1 C (O) NH 2, 35) -NR 1 C (O) NHR ¹ , 36) -NR ¹ C (O) N (R ¹ ) R ¹ , 37) -NHS (O) ₂ R ¹ , 38) -NR ¹ S (O) ₂ R ¹ , 39)- ^{_{S (O) 2 NH 2,}} 40) -S (O) 2 NHR 1, 41) -S (O) 2 N (R 1) R 1, 42) -S (O) R 1, 43) -S ( _{^{O) 2 R 1, 44)}} -OS (O) 2 R 1, 45) -S (O) 2 oR 1, 46) - substituted with benzyl (two or three ^{R a} or ^{R 1} substituent Have May also be), 47)-L-heteroaryl (may be substituted by one or more R ^A or R ¹ substituent, the substituents are attached to either or both of L and heteroaryl groups 48) -L-heterocyclyl (which may be substituted with one or more R ^A or R ¹ substituents, wherein the substituent is attached to either or both of the L and heterocyclyl groups). 49) -L-aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both of the L and aryl groups) _{^{, 50) -L-NR 1 (}} R 1), 51) -L-) 2 NR 1, 52) -L- (n (R 1) -L) n -N (R 1) R 1, 53) - ^{L- (n (R 1) -L} ) n - heteroaryl (one or more ^{R a} or ^{R 1} location May be substituted with a group, the substituents are attached to either or both of L and heteroaryl ^{groups), 54) -L- (N (} R 1) -L) n - heterocyclyl (1 May be substituted with one or more R ^A or R ¹ substituents, which are attached to either or both of L and heterocyclyl groups), 55) -L- (N (R ¹ ) -L) _n -aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bonded to either or both of the L and aryl groups), 56 ^{) -O-L-N (R} 1) R 1, 57) -O-L- heteroaryl (may be substituted by one or more ^{R a} or ^{R 1} substituent, the substituents L and Bonded to either or both heteroaryl groups), 58) -OL- heterosa Krill (may be substituted by one or more R ^A or R ¹ substituent, the substituents are attached to either or both of L and heterocyclyl group), 59) -O-L -Aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both L and the aryl group), 60) -OL _{^{2 -NR 1, 61) -O-}} L- (n (R 1) -L) n -N (R 1) R 1, 62) -O-L- (n (R 1) -L) n - hetero Aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bonded to either or both L and the heteroaryl group), 63) —OL— _{^{(n (R 1) -L)}} n - is substituted with a heterocyclyl (one or more ^{R a} or ^{R 1} substituent At best, the substituents are attached to either or both of L and heterocyclyl ^{group), 64) -O-L- (} N (R 1) -L) n - aryl (one or more of which may be substituted by ^{R a} or ^{R 1} substituent), 65) -S-L-heteroaryl (optionally substituted with one or more ^{R a} or ^{R 1} substituent), 66 ) -S-L-heterocyclyl (optionally substituted by one or more ^{R a} or ^{R 1} substituent), 67) -S-L- aryl (one or more ^{R a} or R ¹ substituent may be substituted, and the substituent is bonded to either or both of L and aryl group)), 68) -SL) ₂ NR ¹ , 69)-
_{^{S-L- (N (R 1}} ) -L) n -N (R 1) R 1, 70) -S-L- (N (R 1) -L) n - heteroaryl (one or more may be substituted by R ^{a substituents), 71) -S-L- (} n (R 1) -L) n - optionally substituted with heterocyclyl (one or more ^{R a} substituents 72) -SL- (N (R ¹ ) -L) _n -aryl (optionally substituted with one or more ^RA substituents), 73) -NR ¹ (R _{^{1), 74) - (n}} (R 1) -L) n -N (R 1) R 1, 75) -N (R 1) L) 2 -NR 1, 76) - (n (R 1) - _{^{L) n -N (R 1)}} R a, 77) - (n (R 1) -L) n - may be substituted with a heteroaryl (one or more ^{R a} or ^{R 1} substituent) 78)-(N ( R ¹ ) -L) _n -heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents), 79)-(N (R ¹ ) -L) _n -aryl ( 80) -heteroaryl (optionally substituted with one or more R ^A substituents), or 81) optionally substituted with one or more R ^A or R ¹ substituents) -Aryl (optionally substituted with one or more R ^A substituents). In this list, each substituent may be attached to the L group if it is not already present; when two R ¹ substituents are present on the same nitrogen atom, each R ¹ substituent is , Independently selected from the list of R ¹ values described below; n is an integer equal to any of 0, 1, 2, 3, 4, or 5; and (R ¹ ) and R ¹ Are bonded to a nitrogen atom, they together with the nitrogen atom may contain one or more other heteroatoms selected from N, O and S. And the ring may be substituted with ^one or more R ¹ or R ^A.

[0035] L is: 1) -C _1-6 alkyl, 2) -C _2-6 alkenyl, 3) -C _2-6 alkynyl, 4) -C _3-7 cycloalkyl, 5) -C _3-7 Cycloalkenyl, 6) heterocyclyl, 7) -C _1-6 alkyl-C _3-7 cycloalkyl, 8) -C _1-6 alkyl-heterocyclyl, 9) aryl, or 10) heteroaryl. In this list, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl and heteroaryl groups may each independently be substituted with one or two R ^A substituents.

[0036] R ¹ is: 1) -H, 2) -C _1-6 alkyl, 3) -C _2-6 alkenyl, 4) -C _2-6 alkynyl, 5) -C _3-7 cycloalkyl, 6 ) -C _3-7 cycloalkenyl, 7) -C _1-5 perfluorinated alkyl , 8) -heterocyclyl, 9) -aryl, 10) -heteroaryl, 11) -benzyl, or 12) 5- [ (3aS, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl] pentanoyl. In this list, the alkyl, alkenyl, alkynyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each independently 1, 2 or 3 R ^A or R ¹ substituents. May be substituted.

[0037] ^{R 2} _{is: 1) -H, 2) -C} 1-6 ^{alkyl, 3) -SR 1, 4)} -C (O) R 1, 5) -S (O) R 1, 6) - S (O) ₂ R ¹ , 7) -Benzyl (optionally substituted with ¹ , 2 or 3 R ^A or R ¹ substituents), 8) -L-heteroaryl (one or more 9) -L-heterocyclyl (one or more) which may be substituted with a R ^A or R ¹ substituent, and the substituent is bonded to one or both of L and heteroaryl groups). R ^A or R ¹ substituent, which is bonded to either or both of L and heterocyclyl groups), 10) -L-aryl (one or more) may be substituted with the R ^a or R ¹ substituent, the substituents are attached to either one or both of L and aryl groups That), 11) - it may be substituted with a heteroaryl (one or more ^{R A} or ^{R 1} substituent), or 12) - aryl (one or more ^{R A} or ^{R 1} substituent May be substituted). In this list, each substituent may be attached to the L group if it is not already present.

[0038] The ^{R A:} 1) - ^{_{halogen, 2) -CF 3, 3)}} -OH, 4) -OR 1, 5) -L-OH, 6) -L-OR 1, 7) -OCF 3, _{^{8) -SH, 9) -SR 1}} , 10) -CN, 11) -NO 2, 12) -NH 2, 13) -NHR 1, 14) -NR 1 R 1, 15) -L-NH 2, 16) -L-NHR ¹ , 17) -L-NR ⁴ R ¹ , 18) -L-SR ¹ , 1
_{^{9) -L-S (O)}} R 1, 20) -L-S (O) 2 R 1, 21) -C (O) OH, 22) -C (O) OR 1, 23) -C (O _{^{) NH 2, 24) -C (}} O) NHR 1, 25) -C (O) N (R 1) R 1, 26) -NHC (O) R 1, 27) -NR 1 C (O) R 1 ^{, 28) -NHC (O) OR} 1, 29) -NR 1 C (O) OR 1, 30) -OC (O) NH 2, 31) -OC (O) NHR 1, 32) -OC (O) ^{N (R 1) R 1,} 33) -OC (O) R 1, 34) -C (O) R 1, 35) -NHC (O) NH 2, 36) -NHC (O) NHR 1, 37) _{^{-NHC (O) N (R 1}} ) R 1, 38) -NR 1 C (O) NH 2, 39) -NR 1 C (O) NHR 1, 40) -NR 1 C (O) N (R 1 ) ^R 1, 41 _{^{-NHS (O) 2 R 1,}} 42) -NR 1 S (O) 2 R 1, 43) -S (O) 2 NH 2, 44) -S (O) 2 NHR 1, 45) -S (O ) ₂ N (R ¹ ) R ¹ , 46) -S (O) R ¹ , 47) -S (O) ₂ R ¹ , 48) -OS (O) ₂ R ¹ , 49) -S (O) ₂ oR ^1, 50) - a) (CF ₃₎ - benzyl, 51) -N _3, or 52) -C (-N = N. In this list, the benzyl group may be substituted with ¹ , 2 or 3 R ^A or R ¹ substituents.

  [0039] In an embodiment of the invention, the compound has the general formula IIA, IIB, IIC, IVA or VIA shown below. Salts or prodrugs of such compounds are also within the scope of the compounds according to the invention.

[0040] In an embodiment of the present invention according to the compound of general formula IIA above, R ¹ , W and R ² are each as defined above.
[0041] In an embodiment of the present invention by the compounds of the general formula IIB, W and R ² are each as defined above, Het is a heterocycle of 3 to 7-membered, the heterocycle one or more R ¹ or R ^A (as defined above) may be substituted.

[0042] In an embodiment of the invention with a compound of general formula IIC above, W and R ² are each as defined above; R ⁵ and R ⁶ are the same or different and are each independently L (as defined above) Or, together with C, forms a 5- to 7-membered ring optionally containing one or more heteroatoms selected from N, O and S, and the ring is one or A plurality of R ¹ or R ^A may be substituted. In a further embodiment, the ring is a 5-membered ring and the heteroatom is a nitrogen atom. In yet a further embodiment, the ring contains 4 nitrogen atoms. In yet a further embodiment, R ² is benzyl.

[0043] In an embodiment of the invention according to the compound of general formula IVA above, W, L, R ¹ and R ² are each as defined above. M, Li, R ³ and R ⁴ are also as defined above.

[0044] In an embodiment of the present invention by the compounds of the general formula IVA, Z is an _CO 2 Me or 2-methyl -2H- tetrazol-5-yl; ^{R 2} is benzyl, 3-thienylmethyl or 3 - be pyridinylmethyl; and W is ^{NH-L-N (R 1} ) is ^{R 1,} wherein, L is _{C 2-4} alkyl, or ^{R 1} is _{C 1-4} alkyl, or (R ¹ ) and R ¹ together with the nitrogen atom to which they are attached may contain one or more other heteroatoms selected from N, O and S A ring is formed, and the ring may be substituted with ^one or more R ¹ or R ^A.

  [0045] In an embodiment of the invention, the compounds of the invention are compounds # 1-55 depicted in Table 1 below. Salts or prodrugs of such compounds are also within the scope of the compounds according to the invention.

[0046] In a further embodiment of the invention, the compound has the formula depicted in Table 1 below. Salts or prodrugs of such compounds are also within the scope of the compounds according to the invention.
Definition:
[0047] Unless otherwise stated, the following definitions apply:

[0048] The singular forms “a”, “an”, and “the” include corresponding plural references unless the context clearly dictates otherwise.
[0049] As used herein, the term "comprising" shall mean that a list of elements following the word "comprising" is required or required, but other Elements are optional and may or may not be present.

  [0050] As used herein, the term "consisting of" shall mean including, but not limited to, whatever follows the phrase "consisting of". Thus, the phrase “consisting of” indicates that the listed element is required or required and that no other element may be present.

[0051] As used herein, the term "alkyl" is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For _{example, C} 1 -C ₆ in C 1 -C ₆ alkyl is defined to include groups having 1,2, 3,4, 5 or 6 carbons in a linear or branched saturated sequences . Examples of C ₁ -C ₆ alkyl as defined above include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, and the like. .

[0052] As used herein, the term "cycloalkyl" shall mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein. For _{example, C} 3 -C ₇ in C 3 -C ₇ cycloalkyl is defined to include 3, 4, 5, 6 or 7 radicals having carbon monocyclic saturated sequences. Examples of C ₃ -C ₇ cycloalkyl as defined above include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

[0053] As used herein, the term "alkenyl" has the specified number of carbon atoms therein, at least two of which are bonded to each other by a double bond, It shall mean an unsaturated linear or branched hydrocarbon group having any and combinations thereof. For _example, C _C 2 -C ₆ at 2 -C ₆ alkenyl has 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement, at least two of its carbon atoms two Defined to include groups joined together by a heavy bond. Examples of C ₂ -C ₆ alkenyl include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, 1-butenyl, and the like.

[0054] As used herein, the term "alkynyl" refers to an unsaturated linear hydrocarbon group having the specified number of carbon atoms therein, wherein at least two carbon atoms are joined together by a triple bond. Shall mean. For example, C ₂ -C ₄ alkynyl includes a 2, 3 or 4 carbons in the chain, at least two of its carbon atoms is defined to include groups which are bonded together by a triple bond. Examples of such alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl and the like.

[0055] As used herein, the term "cycloalkenyl" is intended to mean a monocyclic unsaturated aliphatic hydrocarbon groups having the specified number of carbon atoms therein. For _{example, C} 3 -C ₇ in C 3 -C ₇ cycloalkenyl is defined to include 3, 4, 5, 6 or 7 radicals having carbon monocyclic sequences. Examples of C ₃ -C ₇ cycloalkenyl as defined above include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

[0056] As used herein, the term "halo" or "halogen" shall mean fluorine, chlorine, bromine or iodine.
[0057] As used herein, the term "haloalkyl" shall mean an alkyl as defined above wherein each hydrogen atom may be sequentially replaced by a halogen atom. Examples of haloalkyl include, but are not limited to, CH ₂ F, CHF ₂ and CF ₃ .

  [0058] As used herein, the term "aryl", alone or in combination with another radical, means a carbocyclic aromatic monocyclic group containing 6 carbon atoms, further aromatic, It may be fused to a second 5- or 6-membered carbocyclic group which may be saturated or unsaturated. Examples of aryl include, but are not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl, tetrahydronaphthyl and the like. The aryl may be connected to another group at an appropriate position on the cycloalkyl ring or aromatic ring.

  [0059] As used herein, the term "heteroaryl" is a monocyclic or bicyclic ring system of up to 10 atoms, wherein at least one ring is aromatic, O, N, and It is meant to contain 1 to 4 heteroatoms selected from the group consisting of S. The heteroaryl can be attached via one of the ring carbon atoms or heteroatoms. Examples of heteroaryl are thienyl, benzimidazolyl, benzo [b] thienyl, furyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, Indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl, isochromyl, isomanazolinyl, isoxazolidyl 4,5-b] -pyridine, tetrazolyl, oxadiazolyl, thiadiazolyl, thie Le and fluorescein derivatives and the like, but not limited thereto.

  [0060] As used herein, the term "heterocycle", "heterocyclic" or "heterocyclyl" contains 3 to 4 heteroatoms selected from the group consisting of O, N and S. It shall mean a 4, 5, 6, or 7 membered non-aromatic ring system. Examples of heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, piperidyl, 3,5-dimethylpiperidyl, pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl and the like. The bond to the ring can be on the ring's nitrogen or carbon atom as described below.

  [0061] As used herein, the term "optionally substituted with one or more substituents" or its equivalent term "optionally substituted with at least one substituent" Means that the event of the situation described thereafter may or may not occur, and the description includes when the event or situation occurs and when it does not occur. The definition shall mean 0-5 substituents.

  [0062] As used herein, the term "subject" or "patient" refers to humans and non-human mammals such as primates, cats, dogs, pigs, cows, sheep, goats, horses, rabbits, rats, It means mouse.

[0063] If the substituent itself is incompatible with the synthetic methods described herein, the substituent can be protected by a suitable protecting group (PG) that is stable to the reaction conditions used in these methods. . The protecting groups can be removed at an appropriate point in the reaction sequence of the method to obtain the desired intermediate or target compound. Suitable protecting groups and methods for protecting and deprotecting various substituents using such suitable protecting groups are well known to those skilled in the art. An example is T.W. Greene and P.M. Wuts, “Protecting Groups in Chemical Synthesis” (4th edition), John Wiley & Sons, NY (2007), the contents of which are incorporated herein by reference in their entirety. Examples of protecting groups used throughout, Fmoc, Bn, Boc, although such CBz and COCF _3, without limitation. In some cases, substituents may be specifically selected to be reactive under the reaction conditions used in the methods described herein. Under these circumstances, the reaction conditions convert the selected substituent to another substituent that is useful for the intermediate compound in the methods described herein or that is the desired substituent for the target compound.

[0064] As used herein, the term "pharmaceutically acceptable salt" shall mean both acid addition and base addition salts.
[0065] As used herein, the term "pharmaceutically acceptable acid addition salt" retains the biological effectiveness and properties of a free base that is not biologically or otherwise harmful. Means salts, which include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid , Formed using oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Is done.

  [0066] As used herein, the term "pharmaceutically acceptable base addition salt" retains the biological effectiveness and properties of a free acid that is not biologically or otherwise harmful. It shall mean salt. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include primary, secondary, and tertiary amines, substituted amines including naturally substituted amines, cyclic amines and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine , Tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine , Salts of piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like, but are not limited thereto.

[0067] The compounds according to the invention, or pharmaceutically acceptable salts thereof, may contain one or more asymmetric centers, chiral axes, and chiral planes, so that enantiomers, diastereomers, and other stereoisomers Isomer can occur. They can be defined by absolute stereochemistry such as (R)-or (S)-, or in the case of amino acids (D)-or (L)-. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)-and (S)-, or (D)-and (L) -isomers can be prepared using chiral synthons or chiral reagents, or reversed phase It can also be resolved using conventional techniques such as HPLC. Racemic mixtures can be prepared and then separated into individual optical isomers, or these optical isomers can be prepared by chiral synthesis. Enantiomers are separated by methods known to those skilled in the art, for example after formation of diastereomeric salts by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer specific reagent. Can be divided. One skilled in the art will appreciate that if the desired enantiomer is converted to another chemical by separation techniques, additional steps are required to form the desired enantiomeric form. Alternatively, specific enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents, or by converting one enantiomer to another by asymmetric transformation.

  [0068] Certain compounds according to the invention may exist as a mixture of epimers. Epimer means a diastereoisomer having the opposite configuration at only one of the two or more stereocenters present in each compound.

[0069] The compounds according to the present invention may exist in zwitterionic form and the present invention includes zwitterionic forms of these compounds and mixtures thereof.
[0070] Furthermore, the compounds according to the invention may also exist in hydrated and anhydrous forms. Hydrates of compounds of any of the formulas described herein are also included. In a further embodiment, the compound according to any of the formulas described herein is a monohydrate. In an embodiment of the present invention, the compounds described herein are about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% Below, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less (by weight) of water is included. In other embodiments, the compounds described herein can be about 0.1% or more, about 0.5% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, Contains about 5% or more, or about 6% or more (by weight) of water.

  [0071] It may be convenient or desirable to prepare, purify, and / or handle the compound in the form of a prodrug. Thus, as used herein, the term “prodrug” relates to a compound that when metabolized (eg, in vivo) yields the desired active compound. Typically, the prodrug is inactive or less active than the desired active compound, but can provide advantageous handling, administration, or metabolic properties. Unless otherwise stated, a reference to a particular compound includes its prodrugs.

[0072] As used herein, the term “EC ₅₀ ” shall mean a concentration that results in a 50% increase in the number of cells of CD34 + CD45RA− as compared to vehicle culture (DMSO).

  [0073] As used herein, the term "hematopoietic stem cell" or "HSC" refers to granulocytes (eg, promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (eg, reticulocytes, erythrocytes). Pluripotency and pluripotency that enable differentiation into functional mature cells such as platelets (eg, megakaryoblasts, platelet-producing megakaryocytes, platelets), and monocytes (eg, monocytes, macrophages) Cell having the ability to regenerate while maintaining (self-replicating ability).

  [0074] HSC is part of the starting cell population. These cells are optionally obtained from the body or organs of the body containing cells of hematopoietic origin. Such sources are unfractionated bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph and spleen. Any of the aforementioned crude or unfractionated blood products can be enriched for cells having characteristics of hematopoietic stem cells by methods known to those skilled in the art.

[0075] As used herein, the term "starting cell population" refers to identifying a cell population containing HSCs taken from one of the various sources described above, as is known in the art. means. The ability to enrich the starting cell population with CD34 + cells means that the cell population is selected based on the presence of the cell surface marker CD34 +. CD34 + cells can be detected and counted using, for example, flow cytometry and fluorescently labeled anti-CD34 antibodies. Furthermore, the starting cell population can be used directly for growth or frozen and stored for use at a later time.

  [0076] During hematopoiesis, HSCs first branch to the precursor stage to the myeloid lineage and lymphoid lineage, and then differentiate into bone marrow stem cells (mixed colony forming cells, CFU-GEMM) and lymph stem cells, respectively. Furthermore, bone marrow stem cells are granulated into erythrocytes through erythroblast burst-forming cells (BFU-E) and erythroid colony-forming cells (CFU-E), into platelets through megakaryocyte colony-forming cells (CFU-MEG), It differentiates into monocytes, neutrophils and basophils via sphere-macrophage colony forming cells (CFU-GM), and into eosinophils via eosinophil colony forming cells (CFU-Eo). On the other hand, lymphoid stem cells differentiate into T cells via T lymphoid progenitor cells and B cells via B lymphoid progenitor cells. These bone marrow stem cells and the various hematopoietic progenitor cells derived from them are distinguished by the nature of the colonies they form on soft agar, semi-solid methylcellulose medium, etc. in the presence of various cytokines.

  [0077] The invention relates to a compound or a compound according to the invention and as defined herein in the manufacture of a medicament for the treatment of a subject (or patient) suffering from a disease listed on the following non-limiting list: Also included is the use of the salt, autologous or allogeneic transplantation or treatment of a subject (or patient) suffering from the above or autoimmune diseases. Examples of malignant blood diseases / disorders and congenital diseases are acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma , Non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, atypical erythropoiesis, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiscot-Aldrich syndrome, congenital metabolic disorders (particularly Gaucher's disease) Although there is, it is not limited to these. There are many examples of immunological diseases that can benefit from transplantation, such as multiple sclerosis, lupus, certain types of arthritis, and severe combined immunodeficiency.

[0078] Accordingly, the present invention includes administering HSCs grown with a compound according to the present invention to a patient suffering from any one of the above disorders / malignancies.
[0079] Further, the described compounds and compositions can be used in the following non-limiting situations: autologous or allogeneic transplantation or treatment of subjects (or patients) suffering from the above disorders or autoimmune diseases. Examples of malignant blood diseases / disorders and congenital diseases are acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma , Non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, true erythropoiesis, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiscot-Aldrich syndrome, congenital metabolic disorders (particularly Gaucher's disease) Although there is, it is not limited to these. There are many examples of immunological diseases that can benefit from transplantation, such as multiple sclerosis, lupus, certain types of arthritis, and severe combined immunodeficiency.

[0080] Accordingly, the present invention includes administering HSCs grown with a compound according to the present invention to a patient suffering from any one of the above disorders / malignancies.
[0081] Also encompassed by the invention are cell populations obtained after expansion using the methods according to the invention and described herein. Both hematopoietic stem cells and progenitor cells can be collected from sources such as adults, umbilical cord blood, fetuses or embryos. Cell proliferation using the methods of the present invention can lead to an increase in the number of progenitor cells useful, for example, to speed up the time to neutrophil or platelet engraftment. Such a method involves culturing a starting population comprising HSCs with an agent that can increase the number of HSCs. The starting population can be enriched for the cell surface marker of interest or a combination thereof (eg, CD34 +, CD34 + CD45RA +/−).

[0082] HSC growth method
[0083] Accordingly, the present invention relates to a method of proliferating hematopoietic stem cells, the method comprising: (a) providing a starting cell population comprising hematopoietic stem cells; and (b) suitable for proliferating hematopoietic stem cells ex vivo. Culturing under mild conditions.

[0084] Accordingly, the present invention relates to a method of proliferating hematopoietic stem cells, the method comprising: (a) preparing a starting cell population comprising hematopoietic stem cells; and (b) suitable for proliferating hematopoietic stem cells ex vivo. Culturing under mild conditions.

  [0085] In one particular embodiment, the method for expanding hematopoietic stem cells comprises (a) providing a starting cell population comprising hematopoietic stem cells, and (b) excluding the starting cell population ex vivo. Culturing in the presence of the compound or composition.

  [0086] To prepare a starting cell population, the cell population can first be subjected to an enrichment or purification step that includes negative and / or positive selection of cells based on specific cell markers. Methods for isolating the starting cell population based on specific cell markers interact with fluorescent activated cell sorting (FACS) technology, also called flow cytometry, or specific cell surface markers A solid or insoluble substrate to which an antibody or ligand is bound may be used. For example, the cells are contacted with a solid substrate (eg, a bead column, flask, magnetic particles) containing the antibody and any unbound cells are removed. When a solid substrate comprising magnetic beads or paramagnetic beads is used, the cells bound to the beads can be easily isolated by a magnetic separator.

  [0087] In one embodiment, the starting cell population is enriched in CD34 + cells. Methods for enriching blood cell populations with CD34 + cells include kits sold by Miltenyi Biotec (CD34 + direct isolation kit, Miltenyi Biotec, Bergisch, Gladbach, Germany) or Baxter (Isolex 3000).

  [0088] The amount of umbilical cord blood from single birth is often inadequate for treating adults or older children. One advantage of a growth method using the compounds or compositions of the present invention is that it allows the generation of a sufficient amount of hematopoietic stem cells from only one cord blood unit.

  [0089] Thus, in one embodiment, the starting cell population is derived from neonatal cord blood cells enriched in CD34 + cells. In one related embodiment, the starting cell population is derived from one or two cord blood units.

  [0090] In another embodiment, the starting cell population is derived from human mobilized peripheral blood cells enriched in CD34 + cells. In one related embodiment, the starting cell population is derived from human mobilized peripheral blood cells isolated from a single patient.

[0091] The starting cell population may preferably contain at least 50% CD34 + cells, and in some embodiments more than 90% CD34 + cells.
[0092] The culture conditions of the starting cell population for hematopoietic stem cell expansion will vary depending on the starting cell population, the desired final cell number, and the desired final HSC ratio.

  [0093] In particular, in one particular embodiment using a starting cell population derived from cord blood cells enriched in CD34 + cells, the culture conditions are cytokines and proliferation commonly known in the art of HSC proliferation. Including the use of other cell growth factors such as factors. Such cytokines and growth factors can be biomolecules or small molecules such as IL-1, IL-3, IL-6, IL-11, G-CSF, GM-CSF, SCF, FlT3-L, thrombopoietin (TPO). ), Erythropoietin, and analogs thereof, but are not limited thereto. As used herein, “analog” includes all structural variants of cytokines and growth factors that have biological activities such as natural forms. For example, cytokine receptor agonists such as mutant antibodies with enhanced or reduced biological activity when compared to the natural form or agonist antibodies to the TPO receptor (see, eg, Patent Publication No. WO 2007/145227). Such as, but not limited to, VB22B sc (Fv) 2). The combination of cytokine and growth factor is chosen to grow HSCs and progenitor cells while limiting the production of terminally differentiated cells. In one particular embodiment, the one or more cytokines and growth factors are selected from the group consisting of SCF, Flt3-L and TPO.

[0094] Human IL6 or interleukin-6 (also known as B cell stimulating factor 2) has been reported (Kishimoto, Ann. Review of 1 mm. 23: 1 2005) and is commercially available. Human SCF or stem cell factor (also known as c-kit ligand, mast cell growth factor or Steel factor) has been reported (Smith, MA et al., ACTA Haematologica, 105, 3: 143, 2001).
Are commercially available. Flt3-L or FLT-3 ligand (also called FL) is a factor that binds to the flt3-receptor. This is also reported (Hannum C, Nature
368 (6472): 643-8), which is commercially available. TPO or thrombopoietin (also known as megakaryocyte growth factor (MGDF) or c-Mpl ligand) has been reported (Kaushansky K (2006). N. Engl. J. Med. 354 (19): 2034-45). And are commercially available.

  [0095] The aforementioned chemical and biological components are not only added by adding them to the medium, but also by immobilizing them on the surface of the substrate or support used for cultivation. Specifically, it can also be used by dissolving the components used in a suitable solvent, coating the substrate or support with the resulting solution and then washing away excess components. Such components used may be added to a substrate or support that is pre-coated with a substrate that binds to the components.

[0096] Growth of HSCs can be performed in natural, semi-synthetic, or synthetic media for composition, supplemented with solid, semi-solid, or liquid media for composition, and mixtures of cell growth factors as described above. And any nutrient medium used for culturing hematopoietic stem cells and / or hematopoietic progenitor cells. Such a medium typically contains sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, cytokines, hormones, antibiotics, serum, fatty acids, saccharides and the like. If necessary, other chemical components or biological components may be added to the culture alone or in combination. Such ingredients formulated in the medium include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, Sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various growth factors, etc. Examples of such basal media suitable for HSC growth methods are StemSpan ^™ Serum-Free Expansion Medium (SFEM) (StemCell Technologies, Vancouver, Canada), StemSpan ^™ H3000-Defined Medium (StemScheme CellGro ^™ , SCGM (CellGenix, Freiburg Germany), StemPro ^™ -34 SFM (Invitrogen), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture H12 Mixture F12, McCoy's Medium Medium EMEM), αMEM medium (alpha modified eag Minimal essential medium), RPMI 1640 medium, Isocove's
Modified Dulbecco's Medium (IMDM), StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 (Cambrex), and Stemline II (Sigma-Aldrich), but are not limited thereto.

  [0097] In one embodiment, a compound or composition of the invention is administered during the method of growing said starting cell population under a concentration suitable for HSC growth. In one particular embodiment, the compound or composition is administered at a concentration comprising 1-3000 nmol or such as 1-100 nmol.

  [0098] In one particular embodiment, where the starting cell population consists essentially of CD34 + enriched cells from one or two cord blood units, or from mobilized PB cells, or from harvested bone marrow, the cells are HSCs. Under conditions for expansion, for example 2-21 days, and / or until the indicated fold expansion and characteristic cell population is obtained. In one particular embodiment, the cells are grown ex vivo under conditions for HSC growth under 21 days, 12 days, 10 days, or 7 days.

  [0099] The cell population is then washed to remove any other components of the compound or composition of the invention and / or cell culture, and a suitable cell suspension medium for short-term use, or long-term Resuspended in storage medium, eg, medium suitable for cryopreservation.

[0100] HSCs and / or hematopoietic progenitor cells can be cultured in culture vessels commonly used for animal cell culture, such as Petri dishes, flasks, plastic bags, Teflon ^™ bags. These may be precoated with extracellular matrix or cell adhesion molecules. Such coating materials are collagen I to XIX, fibronectin, vitronectin, laminin 1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteoponin, fibrinogen, various elastins, various proteoglycans, various Cadherin, desmocollin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, alginate gel, It can be a hydrogel, or a fragment thereof. Such a coating material may be a recombinant material having an artificially modified amino acid sequence. Hematopoietic stem cells and / or hematopoietic progenitor cells can be cultured by using a bioreactor that can mechanically control medium composition, pH, etc., and can obtain high-density culture (Schwartz RM, Proc. Natl. Acad. Sci. U.S.A., 88: 6760, 1991; Koller MR, Bone Marlow Transplant, 21: 653, 1998; Koller, MR, Blood, 82: 378, 1993;
G, Bone Marrow Transplant, 35: 1101, 2005).

  [0101] The present invention further provides a cell population having expanded HSCs obtainable or obtained by the above-described proliferation method. In one particular embodiment, such a cell population is resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host, thereby providing a therapeutic composition.

[0102] The present invention further provides cell populations having expanded HSCs or compositions thereof for use in allogeneic or autologous stem cell transplantation in a mammalian subject.
[0103] A subject referred to herein is, for example, a bone marrow donor or an individual at or at risk of having a depleted or limited blood cell level. Optionally, the subject is a bone marrow donor before or after bone marrow collection. The subject may be a bone marrow transplant recipient. The methods described herein can be used for subjects with limited bone marrow reserve, such as elderly subjects or immunodepletion or myeloablative treatments such as chemotherapy for the treatment of leukemia or lymphoma. It is particularly useful for subjects who have received A subject optionally has a reduced blood cell level or is at risk of developing a decrease in blood cell level compared to a control blood cell level. As used herein, the term control blood cell level refers to the average blood cell level in a subject prior to or substantially free of an event that alters the subject's blood cell level. Events that alter a subject's blood cell levels include, for example, anemia, trauma, chemotherapy, bone marrow transplantation and radiation therapy. For example, the subject has anemia or blood loss, eg, due to trauma.

[0104] The transplant may be a composition containing a buffer, an antibiotic, a drug in addition to the hematopoietic stem cells and / or hematopoietic progenitor cells grown by the method of the present invention.
[0105] A composition comprising a proliferating HSC population or a cell population having proliferating HSC is administered to a subject prior to, simultaneously with, or after, for example, chemotherapy, radiation therapy or bone marrow transplantation. The subject optionally has depleted bone marrow associated with, for example, a congenital, hereditary or acquired syndrome characterized by bone marrow loss or depleted bone marrow. Thus, the subject is optionally a subject in need of hematopoiesis. Optionally, the subject is a bone marrow donor or a subject with or at risk of depleted bone marrow.

[0106] Hematopoietic stem cell manipulation is useful as a replacement therapy for chemotherapy or radiation therapy. For example, because HSC is localized in peripheral blood, it is isolated from subjects undergoing chemotherapy and cells are returned after therapy. Thus, a subject is a subject who will or will receive an immune cell depletion treatment such as chemotherapy, radiation therapy, or a subject that serves as a donor for bone marrow transplantation. Because the bone marrow is one of the most proliferating tissues in the body, it is often the first organ damaged by chemotherapeutic drugs and radiation. As a result, the production of blood cells is rapidly destroyed during chemotherapy or radiotherapy, so the chemotherapy or radiation must be discontinued and the hematopoietic system replenished with a supply of blood cells before the patient is retreated with chemotherapy. Don't be. Thus, as described herein, HSC or blood cells produced by the methods described herein may be administered to such patients in need of additional blood cells.

  [0107] A therapeutic agent (eg, small molecule, antibody, etc.) and optionally at least one that can enhance HSC growth in vivo, in vitro, or ex vivo, as described above with an HSC grown by a compound or composition of the invention Provided in combination with one pharmaceutically acceptable excipient or carrier. Therapeutic agents that can enhance HSC proliferation include agonist antibodies to the TPO receptor (eg, VB22B sc (Fv) 2 as detailed in patent publication WO 2007/145227); cytokines, eg, SCF , IL-6, Flt-3 ligand, TPO or TPO mimetics (eg, WO / 2007/022269; WO / 2007/009120; WO / 2004/045415; WO / 2003/103686; WO / 2002/085343; WO / WO / 2001/089457; WO / 2001/039773; WO / 2001/034585; WO / 2001/021180; WO / 2001/021180; WO / 2001/017349; WO / 2000/066112; WO / 20 WO / 2000/028987; WO / 2008/028645; etc.); granulocyte colony stimulating factor (G-CSF); granulocyte macrophage colony stimulating factor (GM-CSF); prostaglandin or prostaglandin reception Body agonists such as prostaglandin E2 receptor-1 (EP-1) agonists, prostaglandin E2 receptor-2 (EP-2) agonists, prostaglandin E2 receptor-3 (EP-3) agonists and Prostaglandin E2 receptor-4 (EP-4) agonist, as detailed in patent publication WO / 2008/073748); tetraethylenepentamine (TEPA); Notch-ligand (Delta-1); Or means a WNT agonist. In addition, culturing stem cells with mesenchymal stem cells (MSCs) can prevent graft-versus-host disease (GVHD) and can help with stem cell proliferation.

  [0108] Pharmaceutically acceptable means a material that is not biologically or otherwise harmful. That is, the material can be administered to a subject or cell without causing undesirable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is contained. The carrier or excipient is chosen to minimize degradation (degradation) of the active ingredient and to minimize adverse side effects in the subject or cells.

  [0109] The compositions are formulated in any conventional manner for use in the methods described herein. Administration is via any route known to be effective by one skilled in the art. For example, the composition is administered transdermally, extracorporeally, intranasally, or topically by oral, parenteral (eg, intravenous), intramuscular injection, by intraperitoneal injection.

[0110] A preferred method of administration is intravenous infusion. The number of cells to be infused takes into account factors such as gender, age, weight, type of disease or disorder, stage of disease, percentage of desired cells in the cell population, and the amount of cells required to produce a therapeutic effect. Put in. In one particular embodiment, the composition is administered by intravenous infusion and, in the case of cord blood, at least ≧ 0.3 × 10 ⁵ CD34 ⁺ / kg or> 2 × 10 ⁶ CD34 ⁺ , bone marrow or mobilized peripheral blood cells In the case, it contains 2.5 × 10 ⁵ CD34 ⁺ / kg or more. In one particular embodiment, all injected cells are derived from proliferating cord blood cells derived from singleton births.

  [0111] The proliferated hematopoietic stem cells and / or hematopoietic progenitor cells, for example, in the case of treatment of leukemia, anticancer agents, whole body irradiation or immunosuppressive agents for eradication of cancer cells or promotion of donor cell engraftment Can be infused by infusion into patients pre-treated with The disease to be treated, pretreatment and cell transplantation method are appropriately selected by the person in charge. The engraftment of hematopoietic stem cells and / or hematopoietic progenitor cells transplanted in this way, the recovery of hematopoiesis, the presence of side effects of transplantation and the therapeutic effect of transplantation can be judged by the usual assays used in transplantation therapy. .

  [0112] As noted above, the present invention allows for the proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells and the use of expanded HSCs to safely and easily perform transplantation therapy in a short period of time. .

[0113] Also provided herein are kits that include one or more containers filled with one or more of the components described herein. Such a kit may contain required or desired solutions and buffers. The kit optionally includes a proliferating stem cell population produced by the above method, or may contain a container or composition for producing a proliferative population of HSC. In particular, the present invention provides a kit for proliferating hematopoietic stem cells ex vivo, the kit comprising a compound as defined in the Summary of the Invention and instructions for using such a compound in an HSC proliferation method; Optionally, one or more cell growth factors, or a medium for cell growth, in particular a medium for HSC growth as described above. The kit may further comprise an antibody for monitoring cell production, for example, an anti-CD34, anti-CD38 and / or anti-CD45RA antibody. In one particular embodiment, such a kit further comprises one or more cell growth factors selected from the group consisting of IL6, FLT3-L, SCF and TPO. Optionally, such a pack or kit is accompanied by instructions for use.

  [0114] In vivo applications: Also provided are kits for providing an effective amount of a compound of the invention for increasing HSC in a subject, the kit comprising one or more of the compounds for use over a period of time. Contains dose. Here, the total number of doses of a compound of the invention in the kit is equal to an effective amount sufficient to increase HSC in the subject. The period is about 1 day to several days or weeks or months. Thus, the duration is at least about 5, 6, 7, 8, 10, 12, 14, 20, 21, 30 or 60 days or any number of days between 1-180 days.

Biological assays screening assays
[0115] To identify novel putative agonists of HSC self-renewal, we adapted a high-throughput screening assay and developed a library of small molecule compounds (5,280 low molecular weight compounds) to primary human mobilization CD34 + Tested against cells. It should be understood that the same approach applies to CD34 + cells from various sources known to those skilled in the art for isolating CD34 + cells. Mononuclear cells were transformed into mouse anti-human CD34 + APC (BD
Pharmingen) followed by magnetic labeling with Anti-APC magnetic microbeads (MACS, Miltenyi Biotec). Magnetically labeled cells were retained using an AutoMACS column. Our study shows that mobilized peripheral blood-derived CD34 + CD45RA-cells cultured in medium supplemented with interleukin-6, thrombopoietin, Flt-3 ligand, and stem cell factor promote mononuclear cell (MNC) proliferation. At the same time, it was based on the fact that it would lead to a decrease in the CD34 + CD45RA− population and HSC depletion. Therefore, low molecular weight compounds that prevent this loss can act as agonists of HSC proliferation.

  [0116] In a 384 well plate, 2000 CD34 + cells / well were cultured in 50 μl medium containing 1 μM test compound or 0.1% DMSO (vehicle). The percentage of CD34 + CD45RA− cells was measured at the start of the experiment and after 7 days of incubation. Six of the 5,280 compounds of different chemical backgrounds initially tested promoted the proliferation of CD34 + CD45RA− cells and 17 as judged by an increased proportion of CD34−CD45RA + cells compared to the control (DMSO). , Enhanced differentiation. Six compounds that promoted proliferation of the CD34 + CD45RA − cell population were reanalyzed in a secondary screen. Four of these six compounds act as aryl hydrocarbon receptor (AhR) antagonists. That is, the mechanism of action has been shown to promote ex vivo growth of huCD34 + cells (same as SR1). The remaining two compounds that were determined not to be aryl hydrocarbon receptor (AhR) antagonists were shown to promote the growth of MNC, including CD34 + cells, during a 7 day incubation. One of these two remaining compounds identified is Compound 1 (Table 1).

[0117] The following biological assays were used to evaluate the effects of the compounds of the invention on hematopoietic stem cell proliferation. Medium: The medium used was the following recombinant cytokines: interleukin-6, thrombopoietin, Flt-3 ligand in the presence of vehicle (DMSO), positive control (SR1), or a compound or compound combination of the present invention. And a serum-free medium supplemented with stem cell factor (final concentration of 100 ng / ml each).
Cell culture: The purity of early harvested CD34 + cells was greater than 90% as measured by flow cytometry. The CD34 + CD45RA− subpopulation reached purity levels higher than 70%. Cells were seeded at 40,000 cells / ml and incubated for 7-12 days at 37 ° C. in 5% CO ₂ . For long-term culture, 200,000 CD34 + cells / ml from mobilized PB are added to interleukin-6, thrombopoietin, Flt3 ligand, and in the presence of vehicle (DMSO), positive control, or 500 nM of a compound of the invention. Culture was performed using serum-free medium supplemented with stem cell factor (final concentration 100 ng / ml each). After 10 days ex vivo culture, Compound 1 (Table 1) increased MNC over 7-fold, CD34 + cell input value (Day 0) over 5-fold, and nearly 4 times the value determined for vehicle. Promoted an increase in After 10 days ex vivo culture, Compound 1 treated cells showed higher levels of CD34 expression (65.8 ± 5.5%) compared to cells cultured with vehicle (DMSO) (22.8 ± 0.9%). ). Furthermore, only Compound 1 treated cells retained the highest expression (24.8 ± 0.9%) of the CD34 + CD45RA− population compared to vehicle (4.7 ± 0.4%). The number of CD34 + CD45RA− cultured with Compound 1 increased about 3 times compared to vehicle and increased 7 times over the input value. Finally, the compounds of the invention were assayed in a dose response format (concentration range 1 nM to 5000 nM) to determine the effective concentration that produced a 50% increase in the number of CD34 + CD45RA− cells compared to vehicle conditions. The results are shown in Table 1.

Compound 1 is not acting through the aryl hydrocarbon (AhR) pathway (FIG. 1)
[0118] We next demonstrated that the effect of Compound 1 on undifferentiated CD34 + CD45RA-undifferentiated hematopoietic cells is rapidly reversible in culture. This effect is best illustrated in FIG. FIG. 2 shows that CD34 + mobilized peripheral blood cells were cultured in the presence of Compound 1 for up to 7 days, at which point the cells were washed to remove Compound 1. The green dotted line indicates that the reduction in the proportion of CD34 + CD45RA− cells is rapidly comparable to that of the control culture (DMSO: solid blue line and dotted line), but cells maintained in the presence of Compound 1 are 2 weeks It retains a more undifferentiated phenotype throughout the culture (green solid line). These results clearly show that within 2 days of compound-free exposure, the cells have acquired a differentiation marker as seen in control cultures. Thus, the effect of Compound 1 is rapidly reversible on undifferentiated human cells.

Compound 1 is not a mitogen (Figure 3)
[0119] We have also shown that Compound 1 does not independently cause cell proliferation in the absence of growth factors. These results are shown in FIG. 3, but as observed with the aryl hydrocarbon receptor antagonist (SR1), compound 1 has three listed growth factors: Flt3, TPO and It indicates that cell proliferation could not be induced in the absence of any of the SCF. This indicates that, similar to SR1, the action of Compound 1 on the maintenance of the undifferentiated HSC phenotype ex vivo is not due to the mitogenic effect on this population but to the prevention of cell differentiation.

Both Compound 1 and Compound 40 prevent cell differentiation and synergize with AhR antagonists (FIG. 4).
[0120] We also evaluated the effect of Compound 1 and one of its potential derivatives, Compound 40, on cell differentiation using both cytological analysis and flow cytometry. Both types of studies showed that Compound 1 and its derivative Compound 40 prevent cell differentiation. The FACS results are shown in FIG. FIG. 4A shows the effect of Compound 1 on mobilized peripheral blood cell differentiation. After 7 days of growth, a relatively pure (> 85% CD34 +) mobilized peripheral blood population was exposed to control (DMSO) or Compound 1 or SR1 or Compound 1 + SR1. The results strongly show that in control cultures cells rapidly lose CD34 cell surface expression, but this effect is partially abolished by introducing optimal levels of SR1 and Compound 1. ing. Interestingly, both SR1 and Compound 1 are synergistic in maintaining CD34 expression on the cell surface. These observations were reproduced both with the cord blood specimen of FIG. 4B and with the compound 40 of FIG. 4C.

[0121] In addition, we have also shown that the effect of Compound 1 or Compound 40 is most impressive on cells with a more undifferentiated phenotype. For example, CD34 + CD45RA− cells (upper left quadrant of the lower panels of FIGS. 4A-4C) are more in cultures supplemented with Compound 1 or 40 than in SR1 or control cultures. The additive effect of Compound 1 or Compound 40 + SR1 has also been observed in these cultures.

  [0122] FIG. 4D provides a dose response curve, which shows that compound 40 is effective in preventing the disappearance of the CD34 marker on the surface of the undifferentiated human HSC-enriched population. Note that CD34 expression varies with different doses of compound.

Compounds 1 and 40 propagate the human HSC phenotype ex vivo (Figure 5)
[0123] FIG. 5 shows that not only Compound 1 but also Compound 40 proliferates the human HSC phenotype ex vivo in short and long term cultures. FIG. 5A shows that the total cell number is increased about 20-fold over the input number in cultures started with CD34 + mobilized peripheral blood (mPB) and maintained for 12 days. The level of proliferation is the same whether the culture is initiated with Compound 1, SR1, Compound 1 + SR1, or control DMSO. Most notably, the effect of Compound 1 is observed on the more undifferentiated CD34 + CD45RA − cell subpopulation, growing about 10-fold in the presence of Compound 1 and about 15-fold in the presence of Compound 1 + SR1. Yes. This observation, together with the results shown in FIG. 4, does not affect Compound 1 on cell proliferation, but rather on the prevention of CD34 + CD45RA− cell differentiation, at the expense of more mature cells ( It strongly suggests that they have resulted in their net growth (without differentiation into mature cells). The results in FIG. 5B show that Compound 1 produces 30-40 fold proliferation of CD34 + CD45RA− cord blood cells over 7 days, but these cells grow about 15 fold in the presence of DMSO (control). . FIG. 5C shows similar results, but this time the culture was extended to 12 days and compound 1 was replaced by compound 40. Again, as shown in the left panel, total cell proliferation is the same whether the cells are exposed to DMSO (control), SR1, compound 40 or compound 40 + SR1. Most impressively, more undifferentiated CD34 + CD45RA− cells proliferated 80-fold over 12 days in cultures supplemented with Compound 40, but these cells were 20-fold in cultures initiated with SR1. It grows less than, indicating the superiority of this compound over aryl hydrocarbon repressor antagonists.

  [0124] In summary, these results show that Compound 1 and Compound 40 have a significant effect on the proliferation of CD34 + and the more undifferentiated CD34 + CD45RA− population, even when using CD34 + derived from mobilized peripheral blood or umbilical cord blood cells. It shows that you are doing.

  [0125] The ex vivo functionality of the proliferating cells was tested using a conventional cultured colony forming unit (CFU-C) assay. Untreated cells or cells incubated with DMSO, positive controls or compounds of the invention were seeded in methylcellulose medium under conventional conditions. As an example, Compound 1 (Table 1, Example 1) expands the number of pluripotent hematopoietic progenitor cells. Methylcellulose cultures of 1000 CD34 + mPB cells treated with Compound 1 for 10 days increased multifold lineage granulocytes, macrophages and megakaryocytes (GEMM colonies) by a 5-fold increase compared to input cells and control cells Resulting in a 10-fold increase. This suggests that Compound 1 described herein also promotes proliferation of pluripotent progenitor cells.

[0126] Effect of Compound 1 on cultured mPB HSCs evaluated using the NSG mouse model (Figure 6)
[0127] We next evaluated the effects of Compound 1 and Compound 40 on cord blood and mobilized peripheral blood human HSCs grown in vitro for 10-12 days and introduced in vivo in the NSG mouse model. The goal of these experiments is to confirm that the effects of our compounds on the undifferentiated HSC phenotype shown in FIGS. 4 and 5 are also observed on long-term repopulated bone marrow stem / progenitor cells . FIG. 6 shows mouse bone marrow reconstitution with human cells evaluated 13 weeks after transplantation. The 50,000 and 500,000 cell outcomes for mobilized blood are shown in 6A. As shown therein, the HSC agonist SR1 was consistently better than DMSO (control) in proliferation of human stem cells as assessed in the NSG mouse model. In these experiments, Compound 1 appears to be superior to SR1. As seen in in vitro cultures, Compound 1 and SR1 also showed a synergistic effect in these experiments (FIG. 6).

Effect of compounds 1 and 40 on cultured umbilical cord blood (CB) HSC evaluated using the NSG mouse model (FIG. 7)
[0128] Figure 7 shows the effect of Compound 1 and Compound 40 on cultured cord blood human HSCs evaluated in vivo in the NSG mouse model. The results in FIG. 7A show that Compound 1 has a clear effect on the reconstitution activity of human cells when compared to the control culture. These experiments were performed in short-term culture, ie 7 days. Most importantly, from FIG. 7B, compound 40 has a very significant effect when using 1500 CD34 + cells, with an average level of reconstitution of 10% compared to 2% for the DMSO control. I understand that. The greater impact of Compound 40 in this experiment than Compound 1 (FIG. 7A) is possibly due to the long culture period used in the experiment described in FIG. 7B. A more definitive in vivo experiment is provided in the next section using a longer culture period (12-16 days).

Effects of Compounds 1 and 40 on cultured cord blood (CB) HSC evaluated using the NSG mouse model (FIG. 8)
[0129] Zandstra et al. Recently documented that a new method called fed-batch optimizes in vitro conditions resulting in human HSC growth (US Pat. No. 7,795,024). We wanted to see if the compounds of the invention were active in these pre-optimized fed-batch conditions. For these studies, we evaluated umbilical cord blood-derived hematopoietic stem cell (HSC) proliferation after 12 or 16 days in vitro culture with fed-batch culture + compound 40. The number of HSCs was evaluated based on the engraftment of human cells in immunodeficient mice.

  [0130] As shown in FIG. 8, the addition of compound 40 (Cpd40) provided a significant effect on the growth of the entire population tested, including CD34 + CD45RA− cells, for at least 16 days. This effect is most impressive at 12-16 days and clearly shows a synergistic effect between fed-batch culture (FB in FIG. 8) and compound 40.

  [0131] In addition, freshly enriched CD34 + cells and cells grown for 12 or 16 days were transplanted into female NOD / SCID / IL-2Rγc-null (NSG) mice. Mice had received sublethal (250 rads) irradiation 24 hours prior to transplantation. Cells were injected intravenously from the tail vein. At defined time points (3 weeks, 9 weeks, and 16 weeks), animals were sacrificed and bone marrow was harvested from 2 tibias and 2 femurs. Bone marrow is depleted of red blood cells and was evaluated by flow cytometry to quantify human cell engraftment. Cells were scored positive for engraftment if ≧ 0.5% of cells were positive for human CD45 and human HLA-ABC. At each time point of evaluation, some animals were sent for independent histological analysis.

  [0132] As shown in Table 2 below, there is an engraftment dose response for each condition at late 16 weeks. Limiting dilution analysis revealed that the highest HSC growth was caused by fed-batch culture + Compound 40 for 12 days (18.4 fold). This was significantly higher than the growth produced by the fed-batch control 12-day culture (8.1 fold). Higher growth was caused by two conditions using a 12 day culture compared to a 16 day culture. These results clearly demonstrate in vitro net human HSC growth in the presence of Compound 40 and its activity under fed-batch conditions (FIG. 8 + Table 2).

[0133] ^* Net HSC proliferation = (#HSC day 0) / (# HSC 12 days or 16 days); measured using limiting dilution analysis (LDA) in NSG mice at 16 weeks
Synthesis method
[0134] The synthetic methods outlined below relate to embodiments of the invention in which the substituent Z is at the 7-position of the pyrimidoindole nucleus. As will be appreciated by those skilled in the art, in the case of embodiments of the invention where the substituent Z is in a different position, such as the 5, 8 or 6 position, especially the 6 position, similar synthetic methods can be used with obvious modifications to the skilled person. Can be implemented.

  [0135] Scheme 1 describes the synthesis of a general precursor (1-VI) to the compounds of the present invention. In the first step, aryl fluoride 1-I is treated with alkyl cyanoacetate 1-II in the presence of a base such as but not limited to sodium hydride. The resulting product 1-III is then treated with a reducing agent such as but not limited to zinc dust in acetic acid to give aminoindole 1-IV, which is treated with formamide and ammonium formate. Convert to pyrimidine 1-V. Compound 1-V is treated with a reagent such as phosphoryl chloride or phosphoryl bromide to give reactive intermediate 1-VI, which is treated with amine 1-VII to give compound 1-VIII of the invention.

[0136] Scheme 2 describes the preparation of compound 2-V. 4,6-dichloro-5-iodopyrimidine 2-II and the corresponding phenol, aniline or thiophenol 2-I, and a base (Morsin M. et al. Chemistry-A European Journal, 2009, vol. 15, # 6, pp. 196 1468-1477) or intermediate 2-III with or without a palladium catalyst. The resulting intermediate 2-III is converted to the tricyclic adduct 2-IV using Pd (OAc) ₂ (Zhang M. et al. Tetrahedron Letters, 2002, vol. 43, p. 8235). Finally, compound 2-V of the invention is obtained according to Example 1 outlined below.

  [0137] Scheme 3: Compound 4-II is treated with hydroxylamine followed by dimethylacetamide dimethyl acetal (Tully WR et al. Journal of Medicinal Chemistry, 1991, vol. 34, p. 2060). This gives compound 5-I.

  [0138] Scheme 4: Intermediate 1B (Example 1 below) was treated with propionitrile in HCl / dioxane followed by basic treatment to give methyl 2-ethyl-4-hydroxy-9H pyrimido [4, 5-b] indole-7-carboxylate (6-I) is provided. The compound 6-II is then obtained according to the procedure described in Example 1.

  [0139] Scheme 5: Starting from methyl 3-fluoro-4-nitrobenzoate 7-I, following the procedure of Example 1, compound 7-II is obtained.

General
[0140] The reported HPLC retention times are for reverse phase HPLC (Agilent, 1200 series) using the following conditions. Solvent A: MeOH: H ₂ O: TFA (5: 95: 0.05); Solvent B: MeOH: H ₂ O: TFA (95: 5: 0.05); Flow rate: 3.0 mL / min; Gradient 2 0 to 100% at 0 min B; Column: Zorbax C18, 3.5 microns, 4.6 × 30 mm: wavelength 220 nm.

[0141] Mass spectra were recorded on an Agilent Technologies 6210 G1969A LC / MSD TOF spectrometer or an Agilent Technologies Quadruple LC / MS Model G6120B using the following LC conditions. Solvent A: AcCN: H ₂ O: HCOOH (5: 95: 0.05); Solvent B: AcCN: H ₂ O: HCOOH (95: 5: 0.05); 0-100% over a gradient of 2.0 minutes B; flow rate: 0.3 mL / min; column: Zorbax C18, 3.5 microns, 2.1 × 30 mm; wavelength 220 nm.

[0142] Experimental procedure
Example 1

Intermediate 1A

[0143] Ethyl 2-cyanoacetate (10.9 mL, 102 mmol) was slowly added to a 60% suspension (0 ° C) of N, N-dimethylformamide (125 mL) in sodium hydride (4.10 g, 102 mmol). A gray suspension was obtained. The mixture was stirred at 0 ° C. for 15 minutes and methyl 4-fluoro-3-nitrobenzoate (10.2 g, 51 mmol) in N, N-dimethylformamide (125 mL) was added. The resulting deep red mixture was stirred at 0 ° C. for 30 minutes and at room temperature for 3 hours. The reaction mixture was diluted with 1N HCl (40 mL) and ethyl acetate (40 mL). The separated aqueous layer was extracted with ethyl acetate (3 × 50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue (26 g). This was purified by flash chromatography (starting with 100% hexanes, gradually adding ethyl acetate increments and complete with 100% ethyl acetate) to give 14.9 g of the title compound. LCMS m / z 291.0 (M−H) ⁻ , retention time (on analytical HPLC) = 1.76 min.

Intermediate 1B

[0144] A 500 mL round bottom flask was charged with methyl 4- (1-cyano-2-ethoxy-2-oxoethyl) -3-nitrobenzoate (14.9 g, 51.0 mmol) and zinc dust (16 mL) in acetic acid (255 mL). 0.7 g, 255 mmol) was added to give a gray suspension. The addition of zinc was carried out over 35 minutes at room temperature under a nitrogen atmosphere and was quite exothermic. The mixture was heated at 100 ° C. for 15 hours. The mixture was allowed to cool, filtered through Celite and rinsed with ethyl acetate. Evaporation gave a residue which was triturated in dichloromethane-hexane and after filtration gave 6.3 g of the title compound. LCMS m / z 263.2 (M + H) ⁺ , retention time (on analytical HPLC) = 1.90 min.

Intermediate 1C

[0145] A 100 mL round bottom flask was charged with 3-ethyl 6-methyl 2-amino-1H-indole-3,6-dicarboxylate (1.1 g, 4.19 mmol), ammonium formate (0.53 g, 8. 39 mmol) and formamide (16.7 mL, 419 mmol) were added to give a brown suspension. This was heated to 165 ° C. for 12 hours. The mixture was allowed to cool to room temperature and water was added. The resulting precipitate was filtered, air dried (air dried) and dried under high vacuum overnight to give 1.1 g of the title compound. LCMS m / z 244.2 (M + H) ⁺ , retention time (on analytical HPLC) = 1.51 min.

Intermediate 1D

[0146] Methyl 4-hydroxy-9H-pyrimido [4,5-b] indole-7-carboxylate (1.1 g, 4.5 mmol) and phosphorus oxychloride (15 mL, 161 mmol) in a 100 mL round bottom flask The mixture was heated to 90 ° C. for 16 hours, cooled to room temperature and evaporated under reduced pressure. The residue was suspended in dichloromethane (20 mL) and filtered through celite. Evaporation gave the title compound as an orange solid (360 mg). LCMS m / z 262.0 (M + H) ⁺ , retention time (on analytical HPLC) = 2.02 min.

Example 1

[0147] A 2-5 mL microwave vial was charged with methyl 4-chloro-9H-pyrimido [4,5-b] indole-7-carboxylate (86 mg, 0.33 mmol), triethylamine (2 mL) in methanol (2 mL). 0.09 mL, 0.66 mmol) and 3- (piperidin-1-yl) propan-1-amine (0.078 mL, 0.49 mmol) were added and the mixture was heated to 140 ° C. in a microwave reactor for 15 minutes. . The mixture was allowed to cool to room temperature and evaporated under reduced pressure. The crude material was dissolved in N, N-dimethylformamide, purified on reverse phase Zorbax SB-C18 column 21.2 × 100 mm and eluted with MeOH-water-0.1% TFA. Gradient: 20% isocratic for 4 minutes, then gradient to 100% MeOH over 15 minutes. The title compound was obtained as the trifluoroacetate salt (the corresponding free base and HCl salt were prepared according to standard procedures known to those skilled in the art). LCMS m / z 368.2 (M + H) ⁺ , retention time (on analytical HPLC) = 1
. 38 minutes.

Example 14

Intermediate 14A

[0148] Intermediate 2A was prepared according to the procedure described in Example 1, starting from 4-fluoro-3-nitrobenzonitrile.
Intermediate 14B

[0149] A 2-5 mL microwave vial was charged with 4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5- in (trifluoromethyl) benzene (2 mL). b] Indole-7-carbonitrile (47.5 mg, 0.142 mmol) and azidotributyltin (409 μl, 1.491 mmol) were added to give a brown suspension. The vial was placed in a microwave and heated to 180 ° C. for 30 minutes. The mixture was concentrated to dryness and MeOH (3 mL) was added followed by 4M HCl in dioxane (1.07 mL, 4.26 mmol) to give a yellow solution. Diethyl ether (3 mL) was added to the resulting solution, and the mixture was stirred at 20 ° C. for 16 hr. The resulting solid was collected on a Buchner. The cake was washed with diethyl ether (3 × 1 mL) and hexane (3 × 1 mL) and the solid was dried to constant weight at 30 ° C. under high vacuum to give 56 mg of the title compound as the HCl salt. LCMS m / z 378.2 (M + H) ⁺ , retention time (on analytical HPLC) = 1.30 min.

Example 14

[0150] To a 25 mL round bottom flask was added tetrahydrofuran (2 mL) and methanol (N- (3- (piperidin-1-yl) propyl) -7- (2H-tetrazol-5-yl) -9H in 0.5 mL). -Pyrimido [4,5-b] indole-4-amine hydrochloride (43 mg, 0.10 mmol) and N, N-diisopropylethylamine (36 μl, 0.21 mmol) were added to give a brown suspension. 2M trimethylsilyldiazomethane in 260 μl, 0.52 mmol) was added, and the resulting thin yellow suspension was stirred for 3 hours at 20 ° C. and acetic acid (59 μl, 1.04 mmol) was added. Continuing for a minute, the solvent was removed in vacuo to give a residue which was purified by flash chromatography (starting with 100% dichloromethane, Add a mixture of dichloromethane: methanol: 28% wt.ammonium hydroxide aqueous solution (90: 10: 1) and add 100% dichloromethane: methanol: 28% wt.ammonium hydroxide aqueous solution (90: 10: 1) The first elution product obtained was 7- (2-methyl-2H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4 , 5-b] indole-4-amine (19 mg) LCMS m / z 392.2 (M + H) ⁺ , retention time (on analytical HPLC) = 1.44 min. The product is 7- (1-methyl-1H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b] indole-4. .LCMS were of amine (5mg) m / z 392.2 ( M + H) +, retention time (analytical on HPLC) = 1.27 min.

Example 15

[0151] NaH (3.41 g, 85 mmol) was added in small portions to a cold solution of 2-cyanoacetamide (7.18 g, 85 mmol) in DMF (53 mL). After 30 minutes at room temperature, 15 mL of methyl 4-fluoro-3-nitrobenzoate (8.5 g, 42.7 mmol)
The solution in DMF was added dropwise. After 3 hours, a mixture of ice, water and 12 mL HCl (10%) was added. The resulting solid was filtered, rinsed with water, and dried overnight under high vacuum to give 9.1 g of methyl 4- (2-amino-1-cyano-2-oxoethyl) -3-nitrobenzoate. : ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.93 (s, 3 H) 5.78 (s, 1 H) 7.77 (s, 1 H) 7.91 (d, J = 7.83 Hz, 1 H) 8.04 ( s, 1 H) 8.39 (dd, J = 8.02, 1.76 Hz, 1H) 8.56 (d, J = 1.56 Hz, 1 H).

[0152] Crude cyano-amide (0.5 g, 1.900 mmol) prepared above with iron (III) chloride hexahydrate (1.540 g, 5.70 mmol) and zinc (1.242 g, 19.00 mmol) Of DMF (4.75 mL) and water (4.75 mL) to give a yellow suspension. After exotherm, the mixture was heated to 100 ° C. for 45 minutes, then slowly cooled to 20 ° C. and stirred for 22 hours. The solid was filtered and washed with DMF (3 × 3 mL) and the filtrate was diluted with water (40 mL) with stirring at 0 ° C. The solid was filtered and the cake was washed with water (2 × 5 mL). Solids contain almost no impurities. The aqueous layer was extracted with EtOAc (3 × 50 mL) and the combined organic layers were washed with water (50 mL) then brine (30 mL). The organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to give 287 mg as a brown solid. This was treated with acetone (6 mL) to obtain a solid suspension. This was diluted with hexane (5 mL). The solid was then collected, dried under high vacuum at 40 ° C. until constant weight, and intermediate 15B methyl 2-amino-3-carbamoyl-1H-indole-6-carboxylate (162 mg, 36.6 yield). %) Was obtained as an off-white solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.80 (s, 3 H) 6.62 (br. S., 2 H) 7.04-7.18 (m, 2 H ) 7.53-7.63 (m, 2 H) 7.72 (s, 1 H) 10.80 (s, 1 H); MS m / z 232.2 (M + H) ⁺ ; HPLC about 96%, RT = 1.37 min.

[0153] Intermediate 15B (0.100 g, 0.429 mmol), methyl 2- (pyridin-3-yl) acetate (0.130 g, 0.858 mmol), and 25% wt in MeOH (0.196 mL). A mixture of sodium methoxide in methanol (0.954 mL) was placed in a microwave oven (microwave oven) and heated to 140 ° C. for 45 minutes. After cooling, AcOH (0.050 mL, 0.879 mmol) was added and the resulting slurry was stirred at 20 ° C. for 1 hour. The solid was filtered, washed with MeOH (3 × 0.5 mL), dried to constant weight at 20 ° C. under high vacuum, intermediate 15C: methyl 4-hydroxy-2- (pyridin-3-ylmethyl) -9H -Pyrimido [4,5-b] indole-7-carboxylate (82 mg, 57.2% yield) was obtained as a brown solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm
3.87 (s, 3 H) 4.09 (s, 2 H) 7.34-7.40 (m, 1 H) 7.79 (dt, J = 8.1, 1.8 Hz, 1 H)
7.83 (dd, J = 8.2, 1.2 Hz, 1 H) 7.99 (d, J = 0.8 Hz, 1 H) 8.02 (d, J = 8.2 Hz, 1 H) 8.48 (dd, J = 4.9, 1.4 Hz, 1 H) 8.60 (d, J = 2.0 Hz, 1 H) 12.49 (br. S., 2 H): MS m / z
335.2 (M + H) ⁺ ; HPLC 95.2% at 220 nm 92.8% at 254 nm, RT = 1.42 min.

[0154] POCl ₃ (0.948 mL) of methyl 4-hydroxy-2- (pyridin-3-ylmethyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.050 g, 0.150 mmol) 10.17 mmol) in a vial and heated to 175 ° C. in a microwave oven for 15 minutes. After cooling, the reaction mixture was poured into ice water (19 mL), then basified to pH 8 by slow addition of 50% aqueous NaOH (2.7 mL) and finally diluted with EtOAc (20 mL). The solid is filtered (first crop of chloride), the aqueous layer is extracted with EtOAc (20 mL), the combined organic layers are dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give the desired chloride derivative. An additional harvest of 35 mg was obtained. The combined isolated harvest of methyl 4-chloro-2- (pyridin-3-ylmethyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (53 mg, 100% yield) was Used as is in the process.

[0155] Methyl 4-chloro-2- (pyridin-3-ylmethyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.053 g, 0.150 mmol), 3- (piperidine-1 -Yl) propan-1-amine (0.072 mL, 0.451 mmol) and triethylamine (0.063 mL, 0.451 mmol) in MeOH (2.5 mL) in a vial and placed in a microwave oven for 15 minutes at 140 <0> C. Heated. After cooling and evaporation of the solvent, the residue was purified by flash chromatography to give 23 mg of yellow oil + solid. This was diluted with CH ₃ CN (3 mL) and stirred for 30 minutes. The solid was filtered and washed with CH ₃ CN (2 × 0.5 mL), then dried under high vacuum at 30 ° C. until constant weight, compound of Example 15: methyl 4-((3- (piperidine-1 1-yl) propyl) amino) -2- (pyridin-3-ylmethyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (11 mg, 16% yield) was obtained as a brown solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.31-1.44 (m, 2 H) 1.44-1.56 (m, 4 H) 1.71-1.86 (m, 2 H) 2.17-2.47 (m, 6 H) 3.56 -3.66 (m, 2 H) 3.88 (s, 3 H) 4.08 (s, 2 H) 7.28-7.34 (m, 1 H) 7.43 (t, J = 5.5 Hz, 1 H) 7.76 (dt, J = 7.8 , 2.0 Hz, 1 H) 7.81 (dd, J = 8.2, 1.4 Hz, 1 H) 7.99 (d, J = 1.4 Hz, 1 H) 8.35 (d, J = 8.2 Hz, 1 H) 8.41
(dd, J = 4.7, 1.6 Hz, 1 H) 8.59 (d, J = 2.0 Hz, 1 H) 12.08 (s, 1 H); MS m / z 459.2 (M + H) ⁺ ; HPLC> 99.5 %, RT = 1.43 minutes.

Example 22

[0156] Methyl 2-((benzyloxy) (phenyl) methyl) -4-chloro-9H-pyrimido [4,5-b] indole-7-carboxylate (prepared as described in Example 15, 0 228 g, 0.498 mmol), 3- (piperidin-1-yl) propan-1-amine (0.158 mL, 0.996 mmol) and triethylamine (0.173 mL, 1.245 mmol) in MeOH (3.8 mL). The mixture was placed in a microwave oven and heated to 140 ° C. for 30 minutes. After cooling to room temperature, the mixture is concentrated to dryness and the residue is purified by flash chromatography to give methyl 2-((benzyloxy) (phenyl) methyl) -4-((3- (piperidin-1-yl) propyl) Amino) -9H-pyrimido [4,5-b] indole-7-carboxylate (172 mg, 61.3% yield) was obtained as a pale yellow solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.29-1.42 (m, 2 H) 1.42-1.56 (m, 4 H) 1.72-1.91 (m, 2 H) 2.18-2.47 (m, 6 H) 3.66 (tt, J = 13.2, 6.6 Hz, 2 H ) 3.88 (s, 3 H) 4.54 (d, J = 11.7 Hz, 1 H) 4.64 (d, J = 12.1 Hz, 1 H) 5.50 (s, 1 H) 7.21-7.43 (m, 8 H) 7.51 ( (t, J = 5.9 Hz, 1 H) 7.57 (d, J = 7.0 Hz, 2 H) 7.82 (dd, J = 8.2, 1.2 Hz, 1 H) 8.00 (d, J = 1.2 Hz, 1 H) 8.38 ( d, J = 8.2 Hz, 1 H) 12.22 (s, 1 H); HRMS m / z 564.2979 (M + H) ⁺ ; HPLC 99.6%, RT = 2.02 min.

[0157] Derivatives methyl 2-((benzyloxy) (phenyl) methyl) -4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b] indole-7- Hydrogenolysis of the benzyl group against carboxylate (0.042 g, 0.075 mmol), Pd—C 10% wt. Performed under hydrogen in methanol in the presence of (moisture 50%) (0.159 g, 0.075 mmol) and ammonium formate (0.235 g, 3.73 mmol). After stirring for 26 hours at 55 ° C., the reaction mixture was filtered through celite, rinsed with MeOH and concentrated to dryness on a rotovap to give 133 mg of residue. This was purified by RP HPLC using a Zorbax SB-C18 column 21.2 × 150 mm eluting with MeOH-water-0.1% TFA to yield 21.9 mg (50% yield) of Example 22: Methyl 2- (Hydroxy (phenyl) methyl) -4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxylate TFA salt as a white solid Obtained: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm
1.30-1.43 (m, 1 H) 1.52-1.73 (m, 3 H) 1.79 (br.d, J = 14.5 Hz, 2 H) 1.96-2.09 (m, 2 H) 2.54 (s, 1 H) 2.74- 2.89 (m, 2 H) 3.11 (dt, J = 10.4, 5.4 Hz, 2 H) 3.38 (d, J = 12.1 Hz, 2 H) 3.71-3.77 (m, 2 H) 3.88 (s, 3 H) 5.63 (s, 1 H) 7.19-7.26 (m, 1 H) 7.27-7.35 (m, 2 H) 7.48-7.56 (m, 2 H) 7.62 (t, J = 5.9 Hz, 1 H) 7.85 (dd, J = 8.2, 1.4 Hz, 1 H) 8.03 (d, J = 1.4 Hz, 1 H) 8.38 (d, J = 8.2 Hz, 1 H) 8.92 (br. S., 1 H) 12.21 (s, 1 H) HRMS m / z 474.2511 (M + H) ⁺ ; HPLC> 99%, RT = 1.68 min.

[0158] Dess-Martin periodinane reagent (22.67 mg, 0.053 mmol) was added to methyl 2- (hydroxy (phenyl) methyl) -4-((3- (piperidin-1-yl) propyl) amino) -9H. -Pyrimido [4,5-b] indole-7-carboxylate (compound of Example 22) and TFA (15.7 mg, 0.027 mmol) in DCM (1000 [mu] L, 15.54 mmol) added to a light orange color A solution was obtained. After 1 hour, the solvent was evaporated and the residue was purified by flash chromatography. Example 23: Methyl 2-benzoyl-4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4 , 5-b] indole-7-carboxylate (10 mg, 79% yield) was obtained as a bright yellow solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.27-1.36 (m,
2 H) 1.36-1.48 (m, 4 H) 1.74-1.88 (m, 2 H) 2.14-2.44 (m, 6 H) 3.52-3.68
(m, 2 H) 3.91 (s, 3 H) 7.54 (t, J = 7.8 Hz, 2 H) 7.69 (t, J = 7.4 Hz, 1 H) 7.76 (t,
J = 5.1 Hz, 1 H) 7.90 (dd, J = 8.2, 1.4 Hz, 1 H) 7.94 (d, J = 7.0 Hz, 2 H) 8.10 (d, J = 1.4 Hz, 1 H) 8.52 (d, J = 8.2 Hz, 1 H) 12.43 (s, 1 H); HRMS m / z 472.2342 (M + H) ⁺ ; HPLC 97.1% at 220 nm, 98.9% at 254 nm, RT = 1.86 min.

Example 25

[0159] Hydroxylamine hydrochloride (0.08 g, 1.2 mmol) was added to methyl 4-((3- (piperidin-1-yl) propyl) amino in MeOH (4.00 mL) and pyridine (0.666 mL). ) -2- (3- (2,2,2-trifluoroacetyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (compound of Example 33) (0.285 g, 0 .515 mmol) to give a yellow solution. After heating at 60 ° C. for 5 days, the mixture was concentrated to dryness, the residue was dissolved in DCM (75 mL) and MeOH (15 mL), and the solution was washed with saturated NaHCO ₃ (20 mL). The aqueous layer was extracted twice with a mixture of CH ₂ Cl ₂ (50 mL) and MeOH (10 mL), the combined organic layers were dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give methyl 4-(( 3- (Piperidin-1-yl) propyl) amino) -2- (3- (2,2,2-trifluoro-1- (hydroxyimino) ethyl) benzyl) -9H-pyrimido [4,5-b] Indole-7-carboxylate (293 mg, 100% yield) was obtained as an off-white solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.32-1.43 (m, 2 H) 1.44-1.56 ( m, 4 H) 1.76-1.87 (m, 2 H) 2.23-2.44 (m, 6 H) 3.57-3.67 (m, 2 H) 3.88 (s, 3 H) 4.04-4.12 (m, 2 H) 7.25- 7.33 (m, 1 H) 7.34-7.46 (m, 2 H) 7.50 (m, J = 7.6, 4.1 Hz, 1 H) 7.55 (d, J = 7.4 Hz, 1 H) 7.81 (dd, J = 8.2, 1.2 Hz, 1 H) 7.99 (d, J = 1.2 Hz, 1 H) 8.36 (d, J = 8.2 Hz, 1 H) 12.07 (d, J = 3.5 Hz, 1 H); MS m / z 569.2 (M + H) +; HPLC> 95%, RT = 1.88 min.

  [0160] Tosyl chloride (0.048 g, 0.251 mmol) was added to methyl 4-((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2,2-trifluoro- 1- (hydroxyimino) ethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.130 g, 0.229 mmol), 4-dimethylaminopyridine (2.79 mg, 0.023 mmol) ) And triethylamine (0.038 mL, 0.274 mmol) in small portions to DCM (10.00 mL) to give a white suspension. After 1 hour at room temperature, the amber solution was diluted with DCM (10 mL) and washed with water (3 × 10 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated to dryness to give methyl 4-((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2,2- Trifluoro-1-((tosyloxy) imino) ethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (188 mg, 99% yield) was obtained as a brown foam: MS m / z 723.2 (M + H) +; HPLC> 89%, RT = 2.09 min.

[0161] Methyl 4-((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2,2-trifluoro-1-((tosyloxy)) cooled to -78 ° C A solution of imino) ethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.188 g, 0.260 mmol) in DCM (5.00 mL) was added ammonia (1.689 mL, 78 mmol). ) And the test tube was sealed and warmed to 20 ° C. The reaction mixture turns blue over time and after 3.5 hours is cooled again to −78 ° C. and then gradually warmed to 20 ° C. using a septum + nitrogen outlet to evaporate most of the ammonia. It was. After 3.5 hours, the reaction mixture was filtered on a Buchner to remove most of the ammonium p-toluenesulfonate, the solid was washed with DCM (3 × 1.5 mL), and the filtrate was concentrated to dryness. A yellow foam was obtained. This was purified by flash chromatography to give methyl 4-((3- (piperidin-1-yl) propyl) amino) -2- (3- (3- (trifluoromethyl) diaziridin-3-yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (115 mg, 78% yield) was obtained as a white foam: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.31-1.43 ( m, 2 H) 1.50 (quin, J = 5.3 Hz, 4 H) 1.80 (dt, J = 14.1, 7.0 Hz, 2 H) 2.32 (m, J = 6.7, 6.7 Hz, 6 H) 3.58-3.67 (m , 2 H) 3.88 (s, 3 H) 3.93 (br.d, J = 7.4 Hz, 1 H) 4.05 (br.d, J = 8.6 Hz, 1 H) 4.07 (s, 2 H) 7.32-7.43 ( m, 3 H) 7.47 (m, J = 6.3 Hz, 1 H) 7.59 (s, 1 H) 7.81 (dd, J = 8.4, 1.2 Hz, 1 H) 7.99 (d, J = 1.2 Hz, 1 H) 8.35 (d, J = 8.4 Hz, 1 H) 12.07 (s, 1 H); MS m / z 568.2 (M + H) ⁺ ; HPLC> 94%, RT = 1.72 min.

[0162] Iodine (0.028 g, 0.111 mmol) was added to methyl 4-((3- (piperidin-1-yl) propyl) amino) -2- (3- (3- (trifluoromethyl) diaziridine-3 -Yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.060 g, 0.106 mmol) and triethylamine (0.044 mL, 0.317 mmol) in a mixture of DCM (2 mL). In addition, a yellow solution was obtained. After 15 minutes, the solvent was evaporated under reduced pressure to give a residue. This was purified by flash chromatography to give 95 mg as a yellow foam. The foam was dissolved in DCM (15 mL) and washed with saturated NaHCO 3 (10 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated to dryness to give the compound of Example 25: methyl 4-((3- (piperidin-1-yl) propyl) amino) -2- (3- (3 -(Trifluoromethyl) -3H-diazilin-3-yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (50 mg, 84% yield) was obtained as a pale yellow solid: 1H NMR (400MHz, DMSO-d6) δ ppm 1.36 (m, J = 5.1 Hz, 2 H) 1.48 (quin, J = 5.5 Hz, 4 H) 1.76 (quin, J = 7.0 Hz, 2 H) 2.30 (br .
t, J = 6.5, 6.5 Hz, 6 H) 3.54-3.67 (m, 2 H) 3.88 (s, 3 H) 4.09 (s, 2 H) 7.14 (d, J = 7.8 Hz, 1 H) 7.26 (s , 1 H) 7.38-7.47 (m, 2 H) 7.52 (d, J = 7.4 Hz, 1 H) 7.81
(d, J = 8.2 Hz, 1 H) 7.99 (s, 1 H) 8.35 (d, J = 8.2 Hz, 1 H) 12.06 (s, 1 H); HRMS m / z 566.2497 (M + H) ⁺ ; HPLC 94.5% at 220 nm, 92.9% at 254 nm, RT = 2.05 min.

Examples 33 and 34

[0163] Methyl 2- (3-bromobenzyl) -4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxylate (Examples) CO, aerated in a solution of 15 prepared, 0.090 g, 0.168 mmol), triethylsilane (0.054 mL, 0.336 mmol) and PdCl ₂ (dppf) (6.14 mg, 8.39 μmol), The mixture was heated at 95 ° C. overnight. The crude mixture was purified by preparative HPLC to give 44 mg of the carbonylation product as a solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 1.28-1.40 (m, 1 H) 1.50-1.72 (m, 3 H) 1.73-1.84 (m, 2 H) 1.95-2.06 (m, 2 H) 2.74-2.87 (m, 2 H) 3.03-3.12 (m, 2 H) 3.33-
3.41 (m, 2 H) 3.88 (s, 3 H) 4.20 (s, 2 H) 7.46-7.60 (m, 2 H) 7.73 (d, J = 7.83 Hz, 1 H) 7.79 (d, J = 7.43 Hz , 1 H) 7.84 (dd, J = 8.22, 1.57 Hz, 1 H) 7.91 (s, 1 H) 8.01 (d, J = 1.17 Hz, 1 H) 8.37 (d, J = 8.61 Hz, 1 H) 8.92 (br. s., 1 H) 10.00 (s, 1
H) 12.16 (s, 1 H).

[0164] Trimethyl (trifluoromethyl) silane (0.7 mL, 3.5 mmol) was cooled to 0 ° C. with methyl 2- (3-formylbenzyl) -4-((3- (piperidin-1-yl) Propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.260 g, 0.535 mmol) and cesium fluoride (5.69 mg, 0.037 mmol) were added to a mixture. After stirring at room temperature for 2 days, 2 mL of concentrated HCl in water (0.5 mL) was added and stirred for 15 minutes. The mixture was diluted with ethyl acetate, neutralized with solid Na2CO3, the phases were separated, and the aqueous layer was extracted twice with EA. The combined organic layers were washed with water, dried over anhydrous MgSO4, filtered and the solvent removed to give a residue. This was purified by preparative HPLC to give 126 mg of the corresponding TFA salt: 1H NMR (400 MHz, DMSO-d6) δ ppm 1.24
-1.43 (m, 1 H) 1.49-1.73 (m, 4 H) 1.73-1.82 (m, 2 H) 1.97-2.07 (m, 2 H) 2.80 (q, J = 11.70 Hz, 2 H) 3.02-3.12 (m, 2 H) 3.36-3.42 (m, 2 H) 3.88 (s, 3 H) 4.10 (s, 2 H) 5.12 (q, J = 7.17 Hz, 1 H) 6.81 (br. s., 1 H ) 7.30-7.35 (m, 2 H)
7.36-7.42 (m, 1 H) 7.48-7.58 (m, 2 H) 7.84 (dd, J = 8.41, 1.37 Hz, 1 H) 8.01
(s, 1 H) 8.37 (d, J = 8.61 Hz, 1 H) 8.95 (br. s., 1 H) 12.17 (s, 1 H); MS m / z 554.2 (M + H) +; HPLC RT 2.142 minutes.

[0165] Dess-Martin periodinane (56.5 mg, 0.133 mmol) was added to methyl 4-((3- (piperidin-1-yl) propyl) amino) -2- (3- (2 , 2,2-trifluoro-1-hydroxyethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (20 mg, 0.036 mmol) to give a white suspension. After stirring at 20 ° C. for 1 hour, the mixture was purified by flash chromatography and as Example 33 methyl 4-((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2,2, 2-Trifluoroacetyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (18.4 mg, 92% yield) was obtained as a yellow solid: ¹ H in DMSO-d ₆ NMR was consistent with the desired product but complicated by the presence of the hydrate form; HRMS m / z 554.2384 (M + H) ⁺ ; HPLC> 95%, RT = 1.76 and 1 87 minutes (ketone + hydrate).

Example 35

[0166] A mixture of 4-fluorobenzonitrile (5 g, 41.3 mmol), dibutyltin oxide (2.055 g, 8.26 mmol), and trimethylsilyl azide (8.22 mL, 61.9 mmol) in toluene (165 mL) was added at 100 ° C. And stirred for 16.5 hours. After cooling to room temperature, the organic layer was extracted with NaOH 1M (83 mL) and the aqueous layer was washed with EtOAc (2 × 85 mL). The aqueous layer was acidified to pH 2 with HCl 2M (41.3 mL). The aqueous mixture was extracted twice with EtOAc (200 mL, then 100 mL) and the combined organic layers were washed with brine (60 mL), dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give intermediate 35A (5- (4-fluorophenyl) -2H-tetrazole, 6.61 g, 98% yield) was obtained as a white solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 7.42-7.53 (m, 2 H) 8.04-8.14 (m, 2 H); MS m / z 165.2 (M + H) ⁺ ; HPLC> 99.5%, RT = 1.96 min.

[0167] 5- (4-fluorophenyl) -2H- tetrazole _{(6.61g, 40.3mmol), K 2} CO 3 (6.68g, 48.3mmol), and iodomethane (3.02 mL, 48.3 mmol) Of acetonitrile in acetonitrile (115 mL) was heated to reflux (about 82 ° C.) for 1 hour. After cooling, the mixture was concentrated to dryness and the residue was partitioned between water (75 mL) and EtOAc (100 mL). The layers were separated, the aqueous layer was back extracted with EtOAc (50 mL), and the combined organic layers were washed with water (50 mL) and brine (50 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated to give 9.5 g as a colorless oil. This solidified on standing. The residue was purified by flash chromatography to give two main products: Intermediate 35B as N2 isomer: 5- (4-fluorophenyl) -2-methyl-2H-tetrazole (5.09 g, yield) 70.9%) as a white solid: NOE was not observed between 4.42 ppm methyl group and aromatic proton; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 4.42 (s, 3 H) 7.33-7.45 (m, 2 H) 8.03-8.14 (m, 2 H); MS m / z
179.2 (M + H) ⁺ ; HPLC> 99.5%, RT = 1.75 min.

N1 isomer: 5- (4-fluorophenyl) -1-methyl-1H-tetrazole (1.87 g, 26.1% yield) as a white solid: 4.16 ppm methyl group and 7.89 The observed NOE between two aromatic protons at −7.97 ppm confirms the structure; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 4.16 (s, 3 H) 7.43-7.53 (m, 2
H) 7.89-7.97 (m, 2 H); MS m / z 179.2 (M + H) ⁺ ; HPLC> 99.5%, RT = 1.29 min.

Intermediates 35C and D

[0169] A solution of intermediate 35B (5- (4-fluorophenyl) -2-methyl-2H-tetrazole, 1 g, 5.61 mmol) in sulfuric acid (16.45 mL, 309 mmol) was cooled to 0 ° C. before fuming. Nitric acid (0.288 mL, 6.17 mmol) was added dropwise. After 2.5 hours, more fuming nitric acid (0.065 mL, 1.403 mmol) was added and the mixture was allowed to warm to 20 ° C. After 5 hours, the mixture was poured into a 2: 1 ice-water mixture (150 mL) resulting in the formation of a white suspension. After 30 minutes, the solid was filtered, washed with water (4 × 10 mL, until the pH of the wash was neutral) and dried under high vacuum at 25 ° C. until constant weight: 5- (4-Fluoro-3 -Nitrophenyl) -2-methyl-2H-tetrazole (1.16 g, 93% yield) as an off-white solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 4.47 (s, 3 H) 7.81 (dd, J = 11.2, 8.8 Hz, 1 H) 8.44 (ddd, J = 8.7, 4.2, 2.3 Hz, 1 H) 8.68 (dd, J = 7.2, 2.2 Hz, 1 H); MS m / z 224.2 ( M + H) ⁺ ; HPLC 98.3%, RT = 1.72 min.

[0170] A solution of 2-cyanoacetamide (0.888 g, 10.56 mmol) in DMF (2.268 mL) was added to 60% wt. In mineral oil in DMF (5.67 mL). In addition to the sodium hydride suspension (0.443 g, 11.08 mmol), a gray suspension was obtained. After cooling to 0 ° C. (Note: generation of hydrogen gas), the resulting mixture was stirred at 0 ° C. for 30 minutes. Next, a solution of 5- (4-fluoro-3-nitrophenyl) -2-methyl-2H-tetrazole (1.15 g, 5.15 mmol) in DMF (2.3 mL) was added to give a deep purple solution. . After 3 hours, the reaction mixture was slowly poured into an ice-water mixture (33.0 mL) and concentrated HCl (0.952 mL). Stir the resulting yellow slurry for 30 minutes, filter the solid, wash with water (3 × 5 mL), then hexane (2 × 5 mL), dry under high vacuum at 40 ° C. to constant weight, and add 2-cyano 2- (4- (2-Methyl-2H-tetrazol-5-yl) -2-nitrophenyl) acetamide (1.41 g, 95% yield) was obtained as a yellow solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 4.49 (s, 3 H) 5.77 (s, 1 H) 7.77 (s, 1 H) 7.95 (d, J = 8.2 Hz, 1 H) 8.03 (s, 1 H) 8.51 (dd , J = 8.2, 1.8 Hz, 1 H) 8.70 (d, J = 1.8 Hz, 1 H); MS m / z 288.1 (M + H) ⁺ ; HPLC 96.4% at 220 nm, RT = 1.31 min .

[0171] Iron (III) chloride hexahydrate (2.82 g, 10.44 mmol) and zinc (2.276 g, 34.8 mmol) were combined with 2-cyano-2- (4- (2-methyl-2H- D of tetrazol-5-yl) -2-nitrophenyl) acetamide (1 g, 3.48 mmol)
To the mixture in MF (8.71 mL) and water (8.71 mL) was added in portions to give a yellow suspension. This was heated to 100 ° C. for 1.25 hours. The mixture was then cooled to 20 ° C., diluted with MeOH (50.0 mL), filtered through celite and concentrated under reduced pressure to about 20 mL (to remove most of the MeOH). The mixture was then diluted with water (50 mL) and EtOAc (100 mL), stirred vigorously and filtered. The aqueous layer was extracted with EtOAc (2 × 50 mL) and the combined organic layers were washed with saturated NaHCO ₃ (50 mL) and brine (50 mL). The organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to give 489 mg as a purple solid that was purified by flash chromatography to give 2-amino-6- (2-methyl-2H-tetrazole-5 -Yl) -1H-indole-3-carboxamide (356 mg, 39.7% yield) was obtained as a purple solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 4.38 (s, 3 H) 6.57 (s, 2 H) 7.01 (s, 2 H) 7.61-7.69 (m, 2 H) 7.81 (s, 1 H) 10.77 (s, 1 H); MS m / z 258.2 (M + H) ⁺ ; HPLC About 78%, RT = 1.34 minutes.

[0172] Intermediate 35D (2-amino-6- (2-methyl-2H-tetrazol-5-yl) -1H-indole-3-carboxamide, 0.35 g, 1.361 mmol) in the microwave tube, methyl 2 -25% wt. In phenylacetate (0.288 mL, 2.041 mmol) and MeOH (0.467 mL). A mixture of sodium methoxide and methanol (3.03 mL) was placed in a microwave oven and heated to 140 ° C. for 1 hour. After cooling to room temperature and diluting with water (1 mL) and AcOH (4 mL), the mixture was stirred for 30 minutes to crystallize. The solid was filtered, washed with MeOH (5 × 1 mL), dried under high vacuum at 40 ° C. until constant weight, and 2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H— Pyrimido [4,5-b] indol-4-ol (220 mg, 45.2% yield) was obtained as a brown solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 4.03 (s, 2 H) 4.43 (s, 3 H) 7.24-7.29 (m, 1 H) 7.34 (t, J = 7.8 Hz, 2 H) 7.37 -7.43 (m, 2 H) 7.92 (dd,
J = 8.0, 1.4 Hz, 1 H) 8.04-8.10 (m, 2 H) 12.38 (s, 1 H) 12.47 (s, 1 H); MS m / z 358.2 (M + H) ⁺ ; HPLC 82.9 %, RT = 1.89 minutes.

[0173] In a 2-5 mL microwave vial, the crude product 2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b] indole-4- All (0.220 g, 0.616 mmol) and POCl ₃ (3.90 mL, 41.9 mmol) were added to give a brown suspension. The vial was placed in a microwave oven and heated to 175 ° C. for 15 minutes and then allowed to cool. The reaction mixture was then poured into a mixture of water and ice (80 ml) and basified to pH 8 by slow addition of 50% wt NaOH (11 mL) followed by EtOAc (80 mL). Some solid was filtered and the layers were separated. The aqueous layer was extracted with EtOAc (80 mL) and the organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give the corresponding chloro derivative: 2-benzyl-4-chloro-7- (2-methyl- 2H-tetrazol-5-yl) -9H-pyrimido [4,5-b] indole (189 mg, 82% yield) was obtained as a brown solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 4.31 (s, 2 H) 4.46 (s, 3 H) 7.20-7.26 (m, 1 H) 7.28-7.39 (m, 4 H) 8.09 (dd, J = 8.2, 1.2 Hz, 1 H) 8.21-8.25 (m, 1 H) 8.39 (d, J = 8.2 Hz, 1 H) 12.93 (s, 1 H); MS
m / z 376.2 (M + H) ⁺ ; HPLC 95.6%, RT = 2.30 min.

[0174] 2-Benzyl-4-chloro-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b] indole (0.050 mg) prepared as described above , 0.133 mmol) and _Et 3 N _{(0.037mL, 0.266mmol)} and 3- (piperidin-1-yl) propan-1-amine (0.033 mL, 0.200 mmol) in MeOH (0.6 mL) The mixture was heated in a microwave oven at 140 ° C. for 25 minutes. After cooling and evaporation of the solvent, the residue was purified by RP-HPLC (MeOH-water (0.5% TFA) 20% -100% MeOH), 55 mg of Example 35: 2-Benzyl-7- (2-methyl -2H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b] indole-4-amine 2,2,2-trifluoroacetate ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.27-1.41 (m, 1 H) 1.53-1.72 (m, 3 H) 1.79 (d, J = 13.69 Hz, 2 H) 1.98-2.09 (m, 2 H)
2.75-2.87 (m, 2 H) 3.09 (dt, J = 10.27, 5.23 Hz, 2 H) 3.39 (d, J = 11.35 Hz, 2 H)
3.69 (q, J = 5.87 Hz, 2 H) 4.09 (s, 2 H) 4.44 (s, 3 H) 7.18-7.24 (m, 1 H) 7.31 (t, J = 7.63 Hz, 2 H) 7.35-7.42 (m, 2 H) 7.51 (br. s., 1 H) 7.93 (dd, J = 8.22, 1.17 Hz, 1 H) 8.11 (d, J = 1.17 Hz, 1 H) 8.43 (d, J = 8.22 Hz , 1 H) 9.04 (br. S., 1 H)
12.15 (s, 1 H); HPLC 99% at 254 nm, Rt 2.063 min; HRMS m / z 482.2817 (M + H) ⁺ .

Example 36 (Cyanide to methyloxadiazole)

[0175] Hydroxylamine hydrochloride (32.7 mg, 0.471 mmol) was added to Example 37 (2-benzyl-4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5 -B] Indole-7-carbonitrile, 50 mg, 0.118 mmol) in EtOH (1.5 mL) was added followed by DIPEA (84 μL, 0.483 mmol) to give a pale yellow suspension. After stirring at room temperature for 2.5 days and at 75 ° C. for 6 hours, the solvent was evaporated and water (3 mL) was added. And after stirring for 30 minutes, the solid was collected and washed with water (3 × 1 mL), the solid material was dried under high vacuum at 35 ° C. until constant weight, and (Z) -2-benzyl-N′-hydroxy- 4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboximidamide-HCl (53 mg, 0.107 mmol, 91% yield). Obtained as a brown solid; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.58-1.85 (m, 6 H) 1.98-2.10 (m, 2 H) 2.69-2.90 (m, 2 H) 2.94-3.15 (m, 2 H) 3.34-3.46 (m, 2 H) 3.59-3.74 (m, 2 H) 4.05 (s, 2 H) 5.84 (br. s., 2 H) 7.15-7.24 (m, 1 H) 7.29 (t, J = 7.4 Hz, 3 H) 7.37 (d, J = 7.4 Hz, 2 H) 7.55 (dd, J = 8.2, 1.2 Hz, 1 H) 7.72 (d, J = 1.2 Hz, 1 H) 8.25 (d, J = 8.6 Hz, 1 H) 9.47 (d, J = 7.4 Hz, 1 H) 9.56 (s, 1 H) 11.88 (s, 1 H); HRMS m / z 458.2662
(M + H) ⁺ . 95.4% HPLC at 220 nm 97.4% at 254 nm, RT = 1.40 min.

[0176] Acetic anhydride (0.917 mL, 9.72 mmol) was added to (Z) -2-benzyl-N′-hydroxy-4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [ 4,5-b] indole-7-carboximidamide, HCl (0.040 g, 0.081 mmol) was added to give a brown suspension. The mixture was heated to 140 ° C. for 30 minutes by microwave. The solvent was then evaporated and the residue was purified by flash chromatography to yield 36 mg (85% yield) of 1- (2-benzyl-7- (5-methyl-1,2,4-oxadiazole- 3-yl) -4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b] indol-9-yl) ethanone was obtained as a brown solid, which was immediately added to methanol. Dissolved in (2.3 mL) and treated with DBU (0.021 mL, 0.138 mmol). The resulting yellow solution was heated to reflux for 30 minutes and then cooled to 0 ° C. with stirring for 1 hour. The solid was filtered, washed with cold MeOH (2 × 0.5 mL) and dried to constant weight at 40 ° C. under high vacuum, Example 36: 2-Benzyl-7- (5-methyl-1,2, 4-oxadiazol-3-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b] indole-4-amine (20 mg, 51.3% yield) ) Was obtained as a brown solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.38 (m, J = 4.7 Hz, 2 H) 1.50 (quin, J = 5.5 Hz, 4 H) 1.80 (quin, J = 6.9 Hz, 2 H) 2.18-2.45 (m, 6 H) 2.67 (s, 3 H) 3.57-3.70 (m, 2 H) 4.04 (s, 2 H) 7.15-7.22 (m, 1 H) 7.27 (m, J = 7.6, 7.6 Hz, 2 H) 7.34 (t, J = 5.9 Hz, 1 H) 7.36-7.40 (m, 2 H) 7.83
(dd, J = 8.2, 1.4 Hz, 1 H) 8.01 (d, J = 1.4 Hz, 1 H) 8.39 (d, J = 8.2 Hz, 1 H) 12.02 (br. s., 1 H); HRMS m / z 482.2663 (M + H) ⁺ . HPLC 99.3%, RT = 1.79 min.

Example 37

[0177] 2-Amino-6-cyano-1H-indole-3-carboxamide (0.172 g, 0.859 mmol), methyl 2-phenylacetate (0.303 mL, 2.148 mmol) and 30% wt sodium methoxy in MeOH Of orange (0.403 mL, 2.148 mmol) in methanol (2.82 mL) was placed in a microwave tube and heated at 140 ° C. for 45 minutes. Next, fresh methyl 2-phenylacetate (0.151 mL, 1.074 mmol) and 30% wt sodium methoxide in MeOH (0.201 mL, 1.074 mmol) were added and the vial was placed in the microwave again. Heated at 140 ° C. for 45 minutes. Next, after cooling to room temperature, AcOH (0.197 mL, 3.44 mmol) was added and the resulting slurry was stirred at 20 ° C. for 1 hour. The solid was filtered, washed with MeOH (3 × 1 mL), dried to constant weight at 20 ° C. under high vacuum, intermediate 37B: 2-benzyl-4-hydroxy-9H-pyrimido [4,5-b] Indole-7-carbonitrile (182 mg, 70.5% yield) was obtained as a brown solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 4.03 (s, 2 H) 7.22-7.29 (m, 1 H) 7.30-7.36 (m, 2 H) 7.36-7.43 (m, 2 H) 7.58 (dd, J = 8.2, 1.4 Hz, 1 H) 7.82-7.87 (m, 1 H) 8.05 (d, J = 8.2 Hz, 1 H) 12.59 (br. S., 2 H); MS m / z 301.2 (M + H) ⁺ ; HPLC 94.2% at 220 nm, 91.3% at 254 nm; RT = 1.90 min.

[0178] 2-Benzyl-4-hydroxy-9H-pyrimido [4,5-b] indole-7-carbonitrile (Intermediate 37B, 0.180 g, 0.599 mmol) and phosphorus oxychloride (3.63 mL, 39 0.0 mmol) of the red mixture was heated to 95 ° C. and stirred for 16 hours. After concentrating to dryness on a rotary evaporator, the resulting dark red foam was suspended in saturated NaHCO ₃ (10 mL) and stirred for 30 min. The solid was collected, washed with water (3 × 1 mL), dried to constant weight at 40 ° C. under high vacuum, intermediate 37C: 2-benzyl-4-chloro-9H
-Pyrimido [4,5-b] indole-7-carbonitrile (190 mg, 99% yield) was obtained as a brown solid. This was used as such in the next step: MS m / z 319.2 (M + H) ⁺ ;
HPLC 92.0% at 220 nm, 92.3% at 254 nm, RT = 2.28 min.

[0179] 2-Benzyl-4-chloro-9H-pyrimido [4,5-b] indole-7-carbonitrile (Intermediate 37C, 0.190 g, 0.596 mmol), 3- (piperidin-1-yl) A mixture of propan-1-amine (0.142 mL, 0.894 mmol) and triethylamine (0.208 mL, 1.490 mmol) in MeOH (4.50 mL) was heated to 140 ° C. by microwave for 30 minutes. It was then concentrated to dryness to give 346 mg of an orange solid which was purified by flash chromatography to give 176 mg of a yellow solid. This was suspended in ether (7 mL) and stirred at 20 ° C. for 1 hour. The solid was filtered, washed with ether (3 × 1 mL), dried to constant weight at 30 ° C. under high vacuum, 2-benzyl-4-((3- (piperidin-1-yl) propyl) amino)- 9H-pyrimido [4,5-b] indole-7-carbonitrile was obtained as Example 37 (172 mg, 68.0% yield) as a pale yellow solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.30-1.43 (m, 2 H) 1.43-1.57 (m, 4 H) 1.80 (m, J = 5.5 Hz, 2 H) 2.18-2.47 (m, 6 H) 3.62 (q, J = 6.4 Hz) , 2 H) 4.04 (s, 2 H) 7.15-7.22 (m, 1 H) 7.27 (m, J = 7.4, 7.4 Hz, 2 H) 7.33-7.39 (m, 2 H) 7.47 (t, J = 5.7 Hz, 1 H) 7.62 (dd, J = 8.2, 1.2 Hz, 1 H) 7.79 (d, J = 1.2 Hz, 1 H) 8.43 (d, J = 8.2 Hz, 1 H) 12.19 (s, 1 H) ;
HRMS m / z 425.2448 (M + H) ⁺ ; HPLC> 99%, RT = 1.68 min.

Example 43

[0180] In a 2-5 mL microwave vial, methyl 2-benzyl-4-chloro-9H-pyrimido [4,5-b] indole-7-carboxylate (0.100 g, 0.284 mmol), (E ) -1- (4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) but-3-en-1-yl) piperidine (0.113 g, 0.426 mmol) ), Potassium carbonate (0.106 g, 0.768 mmol) and Pd (Ph ₃ P) ₄ (0.05 g, 0.044 mmol) were added. The vial was purged with N ₂ (3 vacuum + refill cycles). DME (2.84 mL) and water (0.398 mL) were added and the vial was flushed with N2 (1 vacuum + refill) and then heated to 110 ° C. with stirring for 24 hours. After cooling, the mixture is concentrated to dryness under reduced pressure and the residue is purified by flash chromatography to give (E) -methyl 2-benzyl-4- (4- (piperidin-1-yl) but-1-ene- 1-yl) -9H-pyrimido [4,5-b] indole-7-carboxylate (55 mg, 0.121 mmol, 42.6% yield) was obtained as a pale yellow solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.41 (s, 2 H) 1.50-1.61 (m, 4 H) 2.30-2.47 (m, 4 H) 2.53-2.59 (m, 2 H) 2.59-2.70 (m, 2 H) 3.91 (s, 3 H) 4.27 (s, 2 H) 7.16-7.24 (m, 1 H) 7.29 (t, J = 7.6 Hz, 2 H) 7.34-7.43 (m, 4 H) 7.88 (dd, J = 8.2, 1.4 Hz, 1 H) 8.07 (d, J = 1.4 Hz, 1 H) 8.41 (d, J = 8.2 Hz, 1 H) 12.48 (s, 1 H); HRMS m / z 455.2442 (M + H) ⁺ ; HPLC 100% at 220 nm 99.4% at 254 nm, RT = 1.84 min.

[0181] (E) -Methyl 2-benzyl-4- (4- (piperidin-1-yl) but-1-en-1-yl) -9H-pyrimido [4,5-b] indole-7-carboxy Rate (20 mg, 0.044 mmol) and Pd—C 10% wt. A mixture of (moisture 50%) (23.41 mg) in MeOH (2 mL) and THF (2 mL) was treated with hydrogen for 17 h. The reaction mixture was diluted with DCM (3 mL), filtered, rinsed with MeOH (2 × 2 mL), then DCM (2 × 2 mL) and concentrated to dryness to give 19 mg as a pale yellow solid. This was purified by flash chromatography to give a white solid (14 mg) that was treated with CH ₃ CN (2 mL). After stirring the white suspension at 20 ° C. for 1 hour, the solid was filtered, washed with CH ₃ CN (1 × 1 mL), dried under high vacuum at 40 ° C. to constant weight, and the compound methyl 2 of Example 43 -Benzyl-4- (4- (piperidin-1-yl) butyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (14.4 mg, 71.7% yield) as a white solid Obtained: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.36 (m, J = 5.5 Hz, 2 H)
1.45 (quin, J = 5.5 Hz, 4 H) 1.57 (quin, J = 7.3 Hz, 2 H) 1.83 (dt, J = 14.9, 7.4 Hz,
2 H) 2.18-2.31 (m, 6 H) 3.20-3.28 (m, 2 H) 3.91 (s, 3 H) 4.26 (s, 2 H) 7.16
-7.22 (m, 1 H) 7.28 (t, J = 7.4 Hz, 2 H) 7.32-7.38 (m, 2 H) 7.90 (dd, J = 8.2, 1.2 Hz, 1 H) 8.09 (d, J = 1.2 Hz, 1 H) 8.25 (d, J = 8.2 Hz, 1 H) 12.48 (br. S., 1 H);
HRMS m / z 457.2598 (M + H) ⁺ ; HPLC> 99.5%, RT = 1.75 min.

Example 44

[0182] Methyl 2-Benzyl-4-chloro -9H- pyrimido [4, 5-b] indole-7-carboxylate _{(0.100g, 0.284mmol), Et 3} N (0.079mL, 0.569mmol) And a mixture of tert-butyl (3-aminopropyl) (methyl) carbamate (0.080 g, 0.426 mmol) in MeOH (1 mL) was heated in a microwave oven at 140 ° C. for 40 minutes. The solvent was removed under reduced pressure and the residue was purified by flash chromatography to give 0.092 mg of crude Boc derivative. This was used as such in the next step. TFA (1.0 ml, 12.98 mmol) was converted to methyl 2-benzyl-4-((3-((tert-butoxycarbonyl) (methyl) amino) propyl) amino) -9H-pyrimido [4,5-b] indole. A cold suspension of -7-carboxylate (0.092 g, 0.183 mmol) was added dropwise and the mixture was allowed to warm to room temperature over 30 minutes. After dilution with toluene, the solvent was removed under reduced pressure and the residue was then diluted with EtOAc to give 85 mg of solid that was used as such in the next step: HRMS m / z 404.2091 (M + H) ⁺ .

[0183] Methyl 2-benzyl-4-((3- (methylamino) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxylate 2,2,2-trifluoroacetate (0 0.020 g, 0.039 mmol), sodium carbonate (8.81 mg, 0.083 mmol), sodium iodide (1.448 mg, 9.66 μmol) and 2- (2- (2-chloroethoxy) ethoxy) ethanol (6. A mixture of 46 μl, 0.044 mmol) was heated at 70 ° C. in acetone (0.2 mL). After 15 hours, a second portion of 2- (2- (2-chloroethoxy) ethoxy) ethanol reagent (6.46 μl, 0.044 mmol) was added and the mixture was again heated at 70 ° C. for 15 hours. After cooling to room temperature, the mixture was diluted with EtOAc, washed with water, dried over anhydrous MgSO ₄ , filtered, and the solvent was evaporated to give a residue that was purified by flash chromatography to give 9 mg of Example 44. Obtained: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.74-1.85 (m, 2 H) 2.25 (br. S., 3 H) 2.55 (br. S., 2 H) 3.32-3.36 ( m, 4 H) 3.39-3.46 (m, 6 H) 3.51 (t, J = 5.87 Hz,
2 H) 3.64 (q, J = 6.52 Hz, 2 H) 3.88 (s, 3 H) 4.04 (s, 2 H) 4.53 (br. S., 1 H) 7.18 (t, J = 7.40 Hz, 1 H ) 7.28 (t, J = 7.63 Hz, 2 H) 7.37 (d, J = 7.04 Hz, 2 H) 7.55 (t, J = 5.28 Hz, 1 H) 7.82 (dd, J = 8.22, 1.17 Hz, 1 H) ) 7.99 (s, 1 H) 8.27 (d, J = 8.22 Hz, 1 H) 12.05 (s, 1 H); HRMS m / z 536.2855 (M + H) ⁺ ; HPLC RT 2.035 min.

Examples 45 and 51

[0184] Methyl 2-benzyl-4-chloro-9H-pyrimido [4,5-b] indole-7-carboxylate in MeOH (2.000 mL, 49.4 mmol) in a 2-5 mL microwave vial. (0.050 g, 0.142 mmol) and N1- (3-aminopropyl) -N1-methylpropane-1,3-diamine (0.115 mL, 0.711 mmol) were added to give a brown suspension. The vial was placed in a microwave and heated to 140 ° C. for 30 minutes. After 30 minutes, the mixture was concentrated to dryness on a rotary evaporator and the residue was purified by flash chromatography and lyophilized from CH ₃ CN to give two separate products: mono- as Example 45 N-alkylated product: methyl 4-((3-((3-aminopropyl) (methyl) amino) propyl) amino) -2-benzyl-9H-pyrimido [4,5-b] indole-7-carboxy Rate (44 mg, 67.2% yield) as a white solid; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.44 (dt, J = 13.8, 6.6 Hz, 2 H) 1.72 (dt, J = 13.7, 6.8 Hz, 2 H) 2.11 (s, 3 H) 2.24-2.30 (m, 2 H) 2.33 (t, J = 6.7 Hz, 2 H) 2.47 (br. S., 2 H) 3.51-3.61 ( m, 2 H) 3.76-3.85 (m, 3 H) 3.97 (s, 2 H) 7.08-7.15 (m, 1 H) 7.17-7.24 (m, 2 H) 7.27-7.34 (m, 2 H) 7.50 ( t, J = 5.3 Hz, 1 H) 7.76 (dd, J = 8.2, 1.6 Hz, 1 H) 7.92 (d, J = 1.6 Hz, 1 H) 8.20 (d, J = 8.2 Hz, 1 H); MS m / z 461.2 (M + H) ⁺ ; HPLC> 99%, RT = 1.63 min.

[0185] Bis-alkylated product as Example 51: Dimethyl 4,4 '-(((methylazanezyl) bis (propane-3,1-diyl)) bis (azanezyl)) bis (2-benzyl- 9H-pyrimido [4,5-b] indole-7-carboxylate (3.7 mg, 6.71% yield) as a pale yellow solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.18 −
1.29 (m, 4 H) 1.79-1.91 (m, 4 H) 2.25 (s, 3 H) 3.58-3.71 (m, 4 H) 3.84 (s, 6 H) 3.97 (s, 4 H) 7.07-7.16 ( m, 2H) 7.22 (t, J = 7.4 Hz, 4 H) 7.29-7.34 (m, 4
H) 7.53 (t, J = 5.3 Hz, 2 H) 7.77 (dd, J = 8.2, 1.4 Hz, 2 H) 7.96 (d, J = 1.4 Hz, 2 H) 8.22 (d, J = 8.2 Hz, 2 H) 12.01 (s, 2H); MS m / z 776.3 (M + H) ⁺ ; HPLC 94.6% at 220 nm 93.8% at 254 nm, RT = 2.01 min.

Example 52

[0186] 2,5-Dioxopyrrolidin-1-yl-5-((3aS, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanoate ( 12.01 mg, 0.035 mmol) was added to methyl 4-((3-((3-aminopropyl) (methyl) amino) propyl) amino) -2-benzyl-9H-pyrimido [4,5-b] indole- A pale yellow solution was obtained by adding 7-carboxylate (Example 45, 15 mg, 0.033 mmol) and triethylamine (6.81 μL, 0.049 mmol) in DMF (750 μL, 9.69 mmol). After stirring at 20 ° C. for 1 hour, the mixture was concentrated to dryness and the residue was purified by flash chromatography to give a pale yellow foam. Suspend the foam in Et ₂ O (1 mL) and stir for 30 min to collect the solid, wash with Et ₂ O (2 × 0.5 mL), dry to constant weight at 20 ° C. under high vacuum. Compound of Example 52: methyl 2-benzyl-4-((3- (methyl (3- (5-((3aS, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d ] Imidazol-4-yl) pentanamido) propyl) amino) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxylate (17 mg, 76% yield) was obtained as a pale yellow solid. : ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.17-1.34 (m, 3 H) 1.36-1.51 (m, 2 H) 1.51-1.64 (m, 3 H) 1.78 (dt, J = 13.5, 6.6 Hz, 2 H) 2.02 (t, J = 7.4 Hz, 2 H) 2.17
(s, 3 H) 2.32 (t, J = 7.0 Hz, 2 H) 2.40 (t, J = 6.7 Hz, 2 H) 2.55 (d, J = 12.3 Hz, 1 H) 2.77 (dd, J = 12.3, 5.1 Hz, 1 H) 2.99-3.11 (m, 3 H) 3.58-3.68 (m, 2 H) 3.88
(s, 3 H) 4.04 (s, 2 H) 4.08 (m, J = 4.9, 4.9, 2.3 Hz, 1 H) 4.26 (dd, J = 7.6, 5.3 Hz, 1 H) 6.34 (s, 1 H) 6.40 (s, 1 H) 7.14-7.22 (m, 1 H) 7.27 (t, J = 7.4 Hz, 2H) 7.37 (d, J = 7.0 Hz, 2 H) 7.54 (t, J = 5.5 Hz, 1 H ) 7.74 (t, J = 5.5 Hz, 1 H) 7.82 (dd, J = 8.2, 1.6 Hz, 1 H) 7.99 (d, J = 1.6 Hz, 1 H) 8.27 (d, J = 8.2 Hz, 1 H) ) 12.05 (s, 1 H); MS m / z 687.3 (M + H) ⁺ ; HPLC> 99.5%, RT = 1.70 min.

Example 53
[0187] Intermediate 53A was prepared from commercially available ethyl 2 (3-tolyl) acetate. It was then converted to intermediate 53B as described in Examples 15, 45 and 47. The azirine moiety was then developed according to the description provided in Example 25. The final process is based on Example 52.

[0188] Ethyl 2 (m-tolyl) acetate (4.8 g, 26.9 mmol), NBS (
A mixture of 5.27 g, 29.6 mmol) and benzoyl peroxide (0.110 g, 0.454 mmol) was refluxed in CCl ₄ (28 mL). After 5 hours, the reaction was cooled to 5 ° C., filtered and the solvent removed. Purification by flash chromatography using ethyl acetate-hexane gave 3.8 g of the corresponding bromobenzyl derivative: ¹ H NMR (400 MHz, DMSO-d6) δ ppm 1.19 (t, J = 6.70 Hz, 3 H ) 3.67 (s, 2 H) 4.09 (q, J = 6.70 Hz, 2 H) 4.69 (s, 2 H) 7.15-7.25 (m, 1 H) 7.27-7.42 (m, 3 H); leave this material as is Used in the next step.

[0189] Ethyl 2- (3- (bromomethyl) phenyl) acetate (8.11 g, 31.5 mmol) and 4-methylmorpholine 4-oxide hydrate (5.54 g, 47.3 mmol) in 1,4-dioxane The mixture in (110 mL) was heated to 100 ° C. for 1.5 hours. After cooling to room temperature, the solvent volume was halved, then diluted with Et ₂ O: EtOAc (1: 1, 120 mL), washed with water (50 mL), and the aqueous layer was extracted with EtOAc (60 mL). The combined organic layers were washed with water (2 × 50 mL) and then brine (50 mL). The organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to give 4.72 g of a pale yellow oil. This was purified by flash chromatography to give 2- (3-formylphenyl) acetate (2.91 g, 48% yield) as a pale yellow oil: ¹ H NMR (400 MHz, DMSO-d6) δ ppm
1.19 (t, J = 7.0 Hz, 3 H) 3.81 (s, 2 H) 4.09 (q, J = 7.0 Hz, 2 H) 7.52-7.65 (m, 2
H) 7.79-7.85 (m, 2 H) 10.00 (s, 1 H); MS m / z 207.2 (M + H) ⁺ ; HPLC 99%, RT = 1.67 min.

[0190] Trimethyl (trifluoromethyl) silane (3.13 mL, 21.20 mmol) was added to ethyl 2- (3-formylphenyl) acetate (2.91 g, 15.14 mmol) and fluorine cooled to 0-5 ° C. Cesium chloride (0.161 g, 1.060 mmol) was added to the mixture in DMF (20.19 mL). After stirring for 1.5 hours, a 1M TBAF solution in THF (15.14 mL) was added. The resulting yellow solution was stirred at 0-5 ° C. and after 30 minutes the mixture was poured into water (150 mL) and extracted with MTBE (1 × 150 mL, then 2 × 100 mL). After the combined organic layers were washed with water (1 × 150 mL, then 1 × 100 mL) and brine (100 mL), the organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to give 3.84 g of a pale orange oil Got as. This was purified by flash chromatography to give ethyl 2- (3- (2,2,2-trifluoro-1-hydroxyethyl) phenyl) acetate (663 mg, 16% yield) as a colorless oil: ¹ H NMR (400 MHz, DMSO-d6) δ ppm 1.17 (t, J = 7.0 Hz, 3 H) 3.68 (s, 2 H) 4.08 (q, J = 7.0 Hz, 2 H) 5.13 (q, J = 7.4 Hz , 1 H) 6.82 (s, 1 H) 7.24-7.31 (m, 1 H) 7.37 (t, J = 7.2 Hz, 3 H);
MS m / z 263.1 (M + H) ^<+> ; HPLC 93.7% at 220 nm, RT = 1.82 min.

[0191] Benzyl bromide (0.330 mL, 2.78 mmol) was added to ethyl 2- (3- (2,2,2-trifluoro-1-hydroxyethyl) phenyl) acetate (0.648 g, 2.471 mmol). And K ₂ CO ₃ (1.059 g, 7.66 mmol) in acetonitrile (17.00 mL) and the mixture was heated to reflux with stirring (75-80 ° C.) for 24 hours. After cooling and concentration to dryness, the residue was purified by flash chromatography, intermediate 53A: ethyl 2- (3- (1- (benzyloxy) -2,2,2-trifluoroethyl) phenyl) acetate (686 mg, 79% yield) was obtained as a colorless oil: MS m / z 353.2 (M + H) ⁺ ; HPLC 99.7%, RT = 2.19 min.

[0192] Intermediate 15B (0.310 g, 1.329 mmol), ethyl 2- (3- (1- (benzyloxy) -2,2,2-trifluoroethyl) phenyl) acetate (0.679 g, 1. 927 mmol) and 30% wt. In intermediate 53A and MeOH (0.524 mL). A mixture of sodium methoxide in methanol (3.23 mL) resulted in a thin brown suspension, which was heated to 140 ° C. in a microwave apparatus for 1 hour. After cooling and dilution with MeOH (0.75 mL) and AcOH (0.167 mL, 2.92 mmol), the resulting suspension was stirred at 20 ° C. for 2 hours. The solid is then collected, washed with MeOH (4 × 0.5 mL), then dried to constant weight at 40 ° C. under high vacuum and methyl 2- (3- (1- (benzyloxy) -2,2 , 2-trifluoroethyl) benzyl) -4-hydroxy-9H-pyrimido [4,5-b] indole-7-carboxylate (399 mg, 57.6% yield) was obtained as a brown solid: ¹ H NMR (400 MHz, DMSO-d6) δ ppm 3.87 (s, 3 H) 4.09 (s, 2 H) 4.45-4.57 (m, 2 H) 5.23 (q, J = 7.2 Hz, 1 H) 7.20-7.29 (m , 5 H) 7.36-7.51 (m, 3 H) 7.52 (s, 1 H) 7.84 (dd, J = 8.2, 1.4 Hz, 1 H) 8.01 (d, J = 1.4 Hz, 1 H) 8.03 (d, J = 8.2 Hz, 1 H) 12.46 (br. S., 1 H) 12.56 (br. S., 1 H); MS m / z 522.2 (M + H) ⁺ ; HPLC 220 nm at 92.7% 254 nm 91.7%, RT = 2.20 minutes.

[0193] Methyl 2- (3- (1- (benzyloxy) -2,2,2-trifluoroethyl) benzyl) -4-hydroxy-9H-pyrimido [4,5-b] indole-7-carboxylate A mixture of (0.395 g, 0.757 mmol) in phosphorus oxychloride (6 mL, 64.4 mmol) was heated to 90 ° C. for 2 hours. Next, after cooling, it was concentrated to dryness to give 650 mg as a brown foam, which was suspended in saturated NaHCO ₃ (15 mL) and stirred for 1 hour. The solid was filtered, washed with water (3 × 2 mL), dried to constant weight at 40 ° C. under high vacuum, methyl 2- (3- (1- (benzyloxy) -2,2,2-trifluoroethyl) (Benzyl) -4-chloro-9H-pyrimido [4,5-b] indole-7-carboxylate (375 mg, 92% yield) was obtained as a brown solid which was used as such in the next step: MS m / z 540.2 (M + H) ⁺ ; HPLC 92%, RT 2.51 min.

[0194] Methyl 2- (3- (1- (benzyloxy) -2,2,2-trifluoroethyl) benzyl) -4-chloro-9H-pyrimido [4,5-b] indole-7-carboxylate (0.375 g, 0.695 mmol) and a mixture of N1- (3-aminopropyl) -N1-methylpropane-1,3-diamine (0.784 mL, 4.86 mmol) in MeOH (9.83 mL, 243 mmol). Heat to 140 ° C. for 30 minutes in a microwave oven. Next, after concentration to dryness under reduced pressure, the resulting brown oil was purified by flash chromatography to give methyl 4-((3-((3-aminopropyl) (methyl) amino) propyl) amino) -2- (3- (1- (Benzyloxy) -2,2,2-trifluoroethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (288 mg, 63.9% yield) Was obtained as a yellow foam: ¹ H NMR (400 MHz, DMSO-d6) δ ppm
1.48 (dt, J = 14.0, 6.9 Hz, 2 H) 1.75 (quin, J = 6.7 Hz, 2 H) 2.14 (s, 3 H) 2.24-2.42 (m, 4 H) 2.52-2.60 (m, 2 H ) 3.61 (q, J = 6.3 Hz, 2 H) 3.88 (s, 3 H) 4.10 (s, 2 H) 4.49 (s, 2 H) 5.17 (q, J = 7.0 Hz, 1 H) 7.18-7.30 ( m, 5 H) 7.31-7.36 (m, 1 H) 7.39 (t, J = 7.6 Hz, 1 H) 7.44-7.53 (m, 2 H) 7.58 (t, J = 5.3 Hz, 1 H) 7.84
(dd, J = 8.2, 1.4 Hz, 1 H) 8.00 (d, J = 1.4 Hz, 1 H) 8.28 (d, J = 8.2 Hz, 1 H); MS m / z 649.3 (M + H) ⁺ ; 97.6% HPLC at 220 nm 95.5% at 254 nm, RT = 1.96 min.

[0195] A solution of di-tert-butyl dicarbonate (0.124 mL, 0.533 mmol) in DCM (1 mL) was added to methyl 4-((3-((3-aminopropyl) (methyl) amino) propyl) amino. ) -2- (3- (1- (benzyloxy) -2,2,2-trifluoroethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.288 g, 0 .444 mmol) and triethylamine (0.074 mL, 0.533 mmol) in DCM (3 mL) and MeOH (2 mL) were slowly added to give a yellow solution. After stirring at 20 ° C. for 45 minutes, the solution was concentrated to dryness to give 373 mg as a yellow foam. This was purified by flash chromatography to yield intermediate 53B methyl 2- (3- (1- (benzyloxy) -2,2,2-trifluoroethyl) benzyl) -4-((3-((3- ((Tert-Butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxylate (306 mg, 92% yield) was obtained as a yellow foam. ¹ H NMR (400 MHz,
DMSO-d6) δ ppm 1.33 (s, 9 H) 1.52 (dt, J = 14.1, 7.0 Hz, 2 H) 1.67-1.81 (m, 2 H) 2.12 (s, 3 H) 2.28 (t, J = 7.2 Hz, 2 H) 2.34 (t, J = 6.8 Hz, 2 H) 2.92 (q, J = 6.7 Hz, 2 H) 3.54-3.67 (m, 2 H) 3.88 (s, 3 H) 4.10 (s, 2 H) 4.49 (s, 2 H) 5.17 (q,
J = 6.8 Hz, 1 H) 6.76 (t, J = 5.5 Hz, 1 H) 7.16-7.30 (m, 5 H) 7.31-7.36 (m, 1 H
) 7.39 (t, J = 7.6 Hz, 1 H) 7.43-7.51 (m, 2 H) 7.53 (t, J = 5.3 Hz, 1 H) 7.83 (dd,
J = 8.4, 1.4 Hz, 1 H) 8.00 (d, J = 1.4 Hz, 1 H) 8.27 (d, J = 8.2 Hz, 1 H) 12.06 (s, 1
H); MS m / z 749.3 (M + H) ⁺ ; HPLC 97.7%, RT = 2.06 min.

[0196] Methyl 2- (3- (1- (benzyloxy) -2,2,2-trifluoroethyl) benzyl) -4-((3-((3-((tert-butoxycarbonyl) amino) propyl ) (Methyl) amino) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.306 g, 0.409 mmol) and Pd-C 10% wt (50% moisture) ( A mixture of 0.304 g, 0.143 mmol) in MeOH (9.92 mL) was treated with hydrogen at 20 ° C. for 22 hours. The reaction mixture was then filtered through celite and the cake was rinsed with MeOH (2 × 10 mL) and DCM: MeOH (1: 1, 2 × 10 mL), then concentrated to dryness on a rotary evaporator to give 226 mg of oil. It was. This was purified by flash chromatography to give methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2, 2,2-trifluoro-1-hydroxyethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (202 mg, 75% yield) was obtained as a white foam: ¹ H NMR ( 400 MHz, DMSO-d6) δ ppm 1.33 (s, 9 H) 1.48-1.62 (m, 2 H) 1.71-1.85 (m, 2 H) 2.16 (s, 3 H) 2.25-2.35 (m, 2 H) 2.39 (t, J = 6.8 Hz, 2 H) 2.86-3.00 (m, 2 H) 3.56-3.70 (m, 2 H) 3.88 (s, 3 H) 4.06 (s, 2 H) 5.01-5.14 (m, 1 H) 6.77 (m, J = 6.3 Hz, 2 H) 7.30 (d, J = 4.7 Hz, 2 H) 7.36-7.42 (m, 1 H) 7.49 (s, 1 H) 7.51-7.57 (m, 1 H) 7.82 (dd, J = 8.2, 1.2 Hz, 1 H) 7.99 (d, J = 1.2 Hz, 1 H) 8.26 (d, J = 8.2 Hz, 1 H) 12.06 (br. S., 1 H) MS m / z 659.2 (M + H) ⁺ ; HPLC> 97%, RT = 1.90 minutes.

[0197] Methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2,2-trifluoro- Of 1-hydroxyethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.200 g, 0.304 mmol) and Dess Martin periodinane reagent (0.567 g, 1.336 mmol) The mixture in DCM (7.50 mL) was stirred at 20 ° C. for 1 hour. After evaporation to dryness, the residue was purified by flash chromatography to give methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- ( 3- (2,2,2-trifluoroacetyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (181 mg, 91% yield) was obtained as a yellow foam: DMSO-d6 ¹ H NMR in consistent with the desired product, but complicated by the presence of the hydrate form: MS m / z 657.3 (M + H) ⁺ ; HPLC 96.0% 254 nm at 220 nm At 95.3%, RT 1.87 and 1.96 (ketone + hydrate) fractions.

[0198] Methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2,2-trifluoroacetyl ) Benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.180 g, 0.274 mmol), hydroxylamine hydrochloride (0.023 g, 0.329 mmol) and pyridine (0.355 mL) Of MeOH (1.9 mL) was heated in a microwave oven to 65 ° C. for 48 hours. After concentration of the reaction mixture to dryness, the residue solution was washed with saturated NaHCO ₃ (15 mL), the organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give methyl 4-((3-(( 3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2,2-trifluoro-1- (hydroxyimino) ethyl) benzyl) -9H -Pyrimido [4,5-b] indole-7-carboxylate (184 mg, 100% yield) was obtained as a pale yellow foam: MS m / z 672.3 (M + H) ⁺ ; HPLC 96.0% at 220 nm 91.4% at 254 nm, RT = 2.01 min.

[0199] Ts-Cl (0.060 g, 0.315 mmol) was added to methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2. -(3- (2,2,2-trifluoro-1- (hydroxyimino) ethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.184 g, 0.274 mmol) , DMAP (3.35 mg, 0.027 mmol) and triethylamine (0.048 mL, 0.342 mmol) in DCM (12 mL) was added in portions to give a brown solution. After 1 hour, the reaction mixture was diluted with DCM (12 mL), washed with water (3 × 12 mL), the organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give methyl 4-((3- ( (3-((tert-Butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2,2-trifluoro-1-((tosyloxy) imino) ethyl) benzyl ) -9H-pyrimido [4,5-b] indole-7-carboxylate (217 mg, 96% yield) was obtained as a brown foam:
MS m / z 826.2 (M + H) ^<+> ; HPLC 93.2% at 220 nm 91.3% at 254 nm, RT = 2.20 min.

[0200] Methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2,2-trifluoro- A solution of 1-((tosyloxy) imino) ethyl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.217 g, 0.263 mmol) in DCM (5.07 mL) was -78. Cool to 0 ° C. and condense ammonia (1.7 mL, 79 mmol) into a sealed tube. The mixture was slowly warmed to 20 ° C. and stirred for 3 hours. After cooling to −78 ° C. again, a septum with a gas outlet was attached to the sealed tube, and the temperature was gradually raised to 20 ° C. to evaporate the ammonia. After 3 hours, the mixture was concentrated to dryness and then purified by flash chromatography to give methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino. ) -2- (3- (3- (Trifluoromethyl) diaziridin-3-yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (139 mg, 79% yield) white Obtained as a foam: ¹ H NMR
(400 MHz, DMSO-d6) δ ppm 1.34 (s, 9 H) 1.52-1.62 (m, 2 H) 1.72-1.89 (m, 2 H) 2.08-2.25 (m, 3 H) 2.30-2.44 (m, 4 H) 2.94 (q, J = 6.4 Hz, 2 H) 3.64 (q, J = 6.5 Hz, 2 H) 3.88 (s, 3 H) 3.93 (d, J = 8.4 Hz, 1 H) 4.04 (d, J = 8.4 Hz, 1 H) 4.08 (s, 2 H) 6.78 (br. S., 1 H) 7.31-7.41 (m, 2 H) 7.44-7.50 (m, 1 H) 7.54 (t, J = 5.5 Hz, 1 H) 7.59 (s, 1 H) 7.83 (dd, J = 8.4, 1.4 Hz, 1 H) 7.99 (d, J = 1.4 Hz, 1 H) 8.27 (d, J = 8.4 Hz, 1 H) 12.07 (s, 1 H); MS m / z 671.4 (M + H) ⁺ ; HPLC 98.6% at 220 nm, 96.4% at 254 nm, RT = 1.91 min.

[0201] Methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (3- (tri Pre-filled with fluoromethyl) diazidin-3-yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (70 mg, 0.104 mmol) and triethylamine (43.6 μL, 0.313 mmol) To a 5 mL light-shielded round bottom flask, iodine (27.8 mg, 0.110 mmol) was added to obtain a pale yellow solution. After stirring at 20 ° C. for 15 minutes, the solvent was evaporated and the residue was purified by flash chromatography to give methyl 4-((3-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) Propyl) amino) -2- (3- (3- (trifluoromethyl) -3H-diazilin-3-yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (65 mg, 0 097 mmol, 93% yield) was obtained as a pale yellow foam: ¹ H NMR (400 MHz, DMSO-d6) δ ppm 1.33 (s, 9 H) 1.53 (dt, J = 13.8, 7.0 Hz, 2 H) 1.70-1.82 (m, 2 H) 2.15 (br. S., 3 H) 2.24-2.34 (m, 2 H) 2.34-2.42 (m, 2 H) 2.87-2.98 (m, 2 H) 3.55-3.66 ( m, 2 H) 3.88 (s, 3 H) 4.10 (s, 2 H) 6.76 (br. s., 1 H) 7.14 (d, J = 8.2 Hz, 1 H) 7.27 (s, 1 H) 7.43 ( t, J = 7.8 Hz, 1 H) 7.49-7.58 (m, 2 H) 7.83 (dd, J = 8.2, 1.4 Hz, 1 H) 7.99 (d, J = 1.4 Hz, 1 H) 8.27 (d, J = 8.2 Hz, 1 H) 12.06 (s, 1 H); MS m / z 669.2 (M + H) ⁺ ; HPLC 97.6% at 220 nm 97.3% at 254 nm , RT = 2.18 minutes.

[0202] Trifluoroacetic acid (0.400 mL, 5.19 mmol) was added to methyl 4-((3
-((3-((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (3- (trifluoromethyl) -3H-diazilin-3-yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (0.064 g, 0.096 mmol) was added to a solution in DCM (4 mL) to give a pale yellow solution. After stirring at 20 ° C. for 30 min, the reaction mixture was diluted with DCM (15 mL), washed with saturated NaHCO ₃ (10 mL), and the aqueous layer was back extracted with DCM (10 mL). The combined organic layers were dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give intermediate 53C: methyl 4-((3-((3-aminopropyl) (methyl) amino) propyl) amino) -2. -(3- (3- (trifluoromethyl) -3H-diazilin-3-yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (45 mg, 83% yield) was pale Obtained as a yellow foam: HRMS m / z 569.2601 (M + H) ⁺ ; HPLC 97.1% at 220 nm 96.9% at 254 nm, RT = 1.96 min.

[0203] 2,5-Dioxopyrrolidin-1-yl-5-((3aS, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanoate ( 29.2 mg, 0.085 mmol) was added to methyl 4-((3-((3-aminopropyl) (methyl) amino) propyl) amino) -2- (3- (3- (trifluoromethyl) -3H- A mixture of diazilin-3-yl) benzyl) -9H-pyrimido [4,5-b] indole-7-carboxylate (45 mg, 0.079 mmol) and triethylamine (16.55 μL, 0.119 mmol) in DMF (750 μL). To give a yellow solution. After stirring at 20 ° C. for 30 minutes, the reaction mixture was concentrated under high vacuum to a light orange oil and the residue was purified by flash chromatography to give 56 mg as a white solid. This was lyophilized from CH ₃ CN and the compound of Example 53: methyl 4-((3- (methyl (3- (5-((3aS, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamido) propyl) amino) propyl) amino) -2- (3- (3- (trifluoromethyl) -3H-diazilin-3-yl) benzyl)- 9H-pyrimido [4,5-b] indole-7-carboxylate (51 mg, 0.064 mmol, 81% yield) was obtained as a white solid: ¹ H NMR (400 MHz, DMSO-d ₆ ).
δ ppm 1.18-1.34 (m, 3 H) 1.35-1.50 (m, 3 H) 1.50-1.64 (m, 3 H) 1.70-1.82 (m, 2 H) 2.02 (t, J = 7.4 Hz, 2 H) 2.15 (s, 3 H) 2.26-2.34 (m, 2 H) 2.37 (t, J = 6.7 Hz, 2 H) 2.55 (d, J = 12.5 Hz, 1 H) 2.77 (dd, J = 12.1, 5.1 Hz , 1 H) 2.99-3.10 (m, 3 H) 3.56-3.66 (m, 2 H) 3.88 (s, 3 H) 4.03-4.14 (m, 1 H) 4.10 (s, 2 H) 4.26 (dd, J = 7.6, 5.3 Hz, 1 H) 6.34 (s, 1 H) 6.39 (s, 1 H) 7.14 (d, J = 7.4 Hz, 1
H) 7.28 (s, 1 H) 7.44 (t, J = 7.8 Hz, 1 H) 7.52 (d, J = 7.8 Hz, 1 H) 7.56 (t, J = 5.3
Hz, 1 H) 7.73 (t, J = 5.5 Hz, 1 H) 7.83 (dd, J = 8.2, 1.4 Hz, 1 H) 8.00 (d, J = 1.4 Hz, 1 H) 8.28 (d, J = 8.2 Hz, 1 H) 12.07 (s, 1 H); HRMS m / z 795.3368 (M + H) ⁺ ; HPLC 95.4% at 220 nm, 96.2% at 254 nm, RT = 2.06 min.

Example 55

[0204] Methyl 2-benzyl-4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxylate (Example 27, 0.030 g) 0.066 mmol) in 2M methylamine in MeOH (10.00 mL) in a sealed tube and heated to 110 ° C. for 66 hours, then the mixture was cooled to 20 ° C., concentrated to dryness and flash chromatographed. To give 28 mg of a colorless oil. This was suspended in ether (2 mL). After stirring the resulting suspension for 2 hours, the solid was collected on a Buchner, the cake was washed with ether (2 × 0.5 mL), the product was dried to constant weight at 40 ° C. under high vacuum, Example 55: 2-Benzyl-N-methyl-4-((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b] indole-7-carboxamide (23 mg, yield) 77%) was obtained as a white solid: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.38 (m, J = 5.1 Hz, 2 H) 1.50 (quin, J = 5.5 Hz, 4 H) 1.80 ( quin, J = 7.0 Hz, 2 H) 2.22-2.41 (m, 6 H) 2.81 (d, J = 4.3 Hz, 3 H) 3.58-3.68 (m, 2 H) 4.03 (s, 2 H) 7.15-7.21 (m, 1 H) 7.27 (m, J = 7.4, 7.4 Hz, 3 H) 7.34-7.40 (m, 2 H) 7.70 (dd, J = 8.2, 1.4 Hz, 1 H) 7.90 (d, J = 1.4 Hz, 1H) 8.27 (d, J = 8.2 Hz, 1 H) 8.46 (q, J = 4.3 Hz, 1 H) 11.96 (s, 1 H); HRMS m / z 457.2708 (M + H) ⁺ ; HPLC 220 nm > 99.5% at 254 nm 98.9%, RT = 1.53 minutes.

[0205] The reported HPLC retention times are for reverse phase HPLC (Agilent, 1200 series) using the following conditions. Solvent A: MeOH: H ₂ O: TFA (5: 95: 0.05); Solvent B: MeOH: H ₂ O: TFA (95: 5: 0.05); Flow rate: 3.0 mL / min; Gradient 2 0 to 100% at 0 min B; Column: Zorbax C18, 3.5 microns, 4.6 × 30 mm: wavelength 220 nm.

[0206] EC ₅₀ is defined as the concentration that results in a 50% increase in the number of CD34 + CD45RA− cells compared to vehicle culture (DMSO). ^* EC ₅₀ : A> 1000 nM; B = 500-1000 nM; C = 250-500 nM; D = 100-250; E = <100 nM; F => 1.3-fold compound showing proliferation.

Ex vivo functional assay
[0207] The ex vivo functionality of the proliferating cells was tested using a conventional cultured colony forming unit (CFU-C) assay. Untreated cells or cells incubated with DMSO, positive controls or compounds of the invention were seeded in methylcellulose medium under conventional conditions. As an example, Compound 1 (Table 1, Example 1) expands the number of pluripotent hematopoietic progenitor cells. Methylcellulose cultures of 1000 CD34 + mPB cells treated with Compound 1 for 10 days increased multi-lineage granulocyte erythrocytes, macrophages and megakaryocytes (GEMM colonies) 5 times more than input cells and compared to control cells This resulted in a 10-fold increase. This suggests that Compound 1 promotes the proliferation of pluripotent progenitor cells.

In vivo functional assay
[0208] CD34 + mPB cells cultured with compounds of the present invention are transplanted into immunodeficient NOD scid gamma (NSG) mice. Results of 2,000,000 and 500,000 CD34 + mPB cells cultured with Compound 1 (Table 1, Example 1) for 10 days or vehicle control conditions were transplanted into NSG mice. Eight weeks after transplantation, the reconstitution of human hematopoietic cells was checked with NSG bone marrow using antibodies against human CD45. Cells treated with Compound 1 but not the vehicle were able to engraft NSG mice. Furthermore, reconstitution of human bone marrow and lymphatic compartments was also confirmed because bone marrow cells were positive for human CD33 + and CD19 +, respectively. These results indicate that CD34 + mPB grown on Compound 1 not only contributes to engraftment, but also retains the regrowth potential of multi-lineage series in vivo.

Compound combinations
[0209] CD34 + mPB cells cultured with compounds of the present invention are transplanted into immunodeficient NOD scid gamma (NSG) mice. Results of 2,000,000 and 500,000 CD34 + mPB cells cultured with Compound 1 (Table 1, Example 1) for 10 days or vehicle control conditions were transplanted into NSG mice. Eight weeks after transplantation, the reconstitution of human hematopoietic cells was checked with NSG bone marrow using antibodies against human CD45. Cells treated with Compound 1 but not the vehicle were able to engraft NSG mice. Furthermore, reconstitution of human bone marrow and lymphatic compartments was also confirmed because bone marrow cells were positive for human CD33 + and CD19 +, respectively. These results indicate that CD34 + mPB grown on Compound 1 not only contributes to engraftment, but also retains the regrowth potential of multi-lineage series in vivo.

[0210] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art in light of it, And it should be understood that they are also within the scope of this discovery and the appended claims.
Aspects of the invention
Aspect 1 General Formula I or II:
[Where:
Z is
1) -P (O) (OR ¹ ) (OR ¹ ),
^{2) -C (O) OR 1} ,
^{3) -C (O) NHR 1} ,
4) —C (O) N (R ¹ ) R ¹ ,
^{5) -C (O) R 1} ,
6) -CN,
7) ^-SR 1,
_{8) -S (O) 2 NH} 2,
_{^{9) -S (O) 2 NHR}} 1,
_{^{10) -S (O) 2 N}} (R 1) R 1,
^{11) -S (O) R 1} ,
_{^{12) -S (O) 2 R}} 1,
13) -L,
14) -Benzyl (optionally substituted with ¹ , 2 or 3 R ^A or R ¹ substituents),
15) -L-heteroaryl ( which may be substituted with one or more R ^A or R ¹ substituents, the substituents being bonded to either or both of the L and heteroaryl groups),
16) -L-heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents, wherein the substituents are attached to either or both of L and heterocyclyl groups; ),
17) -L-aryl ( which may be substituted with one or more R ^A or R ¹ substituents, which are attached to either or both of the L and heteroaryl groups),
18) -heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents), or
19) -Aryl (optionally substituted with one or more R ^A or R ¹ substituents)
Here, each substituent may be bonded to the L group if it is not already present; when (R ¹ ) and R ¹ are bonded to a nitrogen atom, they are taken together with the nitrogen atom. To form a 3- to 7-membered ring which may contain one or more other heteroatoms selected from N, O and S, and the ring contains one or more R Optionally substituted with ¹ or R ^A ;
W is
1) -H,
2) -halogen,
3) ^-OR 1,
4) -L-OH,
5) ^-L-OR 1,
6) ^-SR 1,
7) -CN,
8) -P (O) (OR ¹ ) (OR ¹ ),
9) -NHR ¹ ,
^{10) -N (R 1) R} 1,
11) _-L-NH 2,
12) ^-L-NHR 1,
13) -L-N (R ¹ ) R ¹ ,
14) ^-L-SR 1,
^{15) -L-S (O)} R 1,
_{16) -L-S (O)} 2 R 1,
17) -LP (O) (OR < ¹ > ) (OR < ¹ > ),
^{18) -C (O) OR 1} ,
_{19) -C (O) NH 2} ,
^{20) -C (O) NHR 1} ,
^{21) -C (O) N (} R 1) R 1,
^{22) -NHC (O) R 1} ,
^{23) -NR 1 C (O)} R 1,
^{24) -NHC (O) OR 1} ,
^{25) -NR 1 C (O)} OR 1,
_{26) -OC (O) NH 2} ,
^{27) -OC (O) NHR 1} ,
^{28) -OC (O) N (} R 1) R 1,
^{29) -OC (O) R 1} ,
^{30) -C (O) R 1} ,
_{31) -NHC (O) NH 2} ,
32) -NHC (O) NHR ¹ ,
33) -NHC (O) N (R ¹ ) R ¹ ,
^{34) -NR 1 C (O)} NH 2,
^{35) -NR 1 C (O)} NHR 1,
^{36) -NR 1 C (O)} N (R 1) R 1,
_{^{37) -NHS (O) 2 R}} 1,
_{^{38) -NR 1 S (O)}} 2 R 1,
_{39) -S (O) 2 NH} 2,
_{^{40) -S (O) 2 NHR}} 1,
_{^{41) -S (O) 2 N}} (R 1) R 1,
^{42) -S (O) R 1} ,
_{^{43) -S (O) 2 R}} 1,
_{^{44) -OS (O) 2 R}} 1,
_{^{45) -S (O) 2 OR}} 1,
46) -benzyl (optionally substituted with ¹ , 2 or 3 R ^A or R ¹ substituents),
47) -L-heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bonded to either or both of the L and heteroaryl groups),
48) -L-heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both of the L and heterocyclyl groups) ,
49) -L-aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both of the L and aryl groups),
^{50) -L-NR 1 (R} 1),
51) -L-) ₂ NR ¹ ,
_{^{52) -L- (N (R 1}} ) -L) n -N (R 1) R 1,
^{53) -L- (N (R 1} ) -L) n - may be substituted with a heteroaryl (one or more ^{R A} or ^{R 1} substituent, the substituents either L and heteroaryl groups Or both)
54) -L- (N (R ¹ ) -L) _n -heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being L and heterocyclyl groups , Or both)
^{55) -L- (N (R 1} ) -L) n - aryl (may be substituted by one or more ^{R A} or ^{R 1} substituent, either substituents of L and aryl groups, or Combined to both),
^{56) -O-L-N (} R 1) R 1,
57) -OL-heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bonded to either or both of the L and heteroaryl groups) ,
58) -OL-heterocyclyl ( which may be substituted with one or more R ^A or R ¹ substituents, wherein the substituent is bonded to either or both of the L and heterocyclyl groups; Is)
59) -OL-aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bonded to either or both of the L and aryl groups),
60) -OL) ₂ -NR ¹ ,
_{^{61) -O-L- (N (}} R 1) -L) n -N (R 1) R 1,
^{62) -O-L- (N (} R 1) -L) n - heteroaryl (one or more ^{R A} or may be substituted by ^{R 1} substituent, the substituents L and heteroaryl groups , Or both)
^{63) -O-L- (N (} R 1) -L) n - may be substituted by heterocyclyl (one or more ^{R A} or ^{R 1} substituent, the substituents L and Heterosaikuri Bound to either or both of the
^{64) -O-L- (N (} R 1) -L) n - aryl (optionally substituted by one or more ^{R A} or ^{R 1} substituent),
65) -SL-heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents),
66) -S-L-heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents),
67) -SL-aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bonded to either or both of the L and aryl groups),
_{^{68) -S-L) 2 NR}} 1,
_{^{69) -S-L- (N (}} R 1) -L) n -N (R 1) R 1,
70) -SL- (N (R _< ¹ _> ) - L) _n -heteroaryl ( optionally substituted with one or more ^RA substituents),
71) -SL- (N (R _< ¹ _> ) - L) _n -heterocyclyl (optionally substituted with one or more ^RA substituents),
72) -SL- (N (R _< ¹ _> ) - L) _n -aryl ( optionally substituted with one or more ^RA substituents),
^{73) -NR 1 (R 1)} ,
_{^{74) - (N (R 1}} ) -L) n -N (R 1) R 1,
_{^{75) -N (R 1) L}} ) 2 -NR 1,
_{^{76) - (N (R 1}} ) -L) n -N (R 1) R A,
77)-(N (R ¹ ) -L) _n -heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents),
78)-(N (R ¹ ) -L) _n -heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents),
^{79) - (N (R 1} ) -L) n - aryl (in one or more of ^{R A} or ^{R 1} substituent may be substituted),
80) -heteroaryl (optionally substituted with one or more R ^A substituents), or
81) -aryl (optionally substituted with one or more R ^A substituents)
Where each substituent may be attached to the L group, if it is not already present,
And when two R ¹ substituents are present on the same nitrogen atom, each R ¹ substituent is independently selected from the list of R ¹ values described below ,
N is an integer equal to 0, 1, 2, 3, 4, or 5;
And when (R ¹ ) and R ¹ are bonded to a nitrogen atom, they together with the nitrogen atom contain one or more other heteroatoms selected from N, O and S May form a 3- to 7-membered ring, which ring may be substituted with ^one or more R ¹ or R ^A ;
L is
1) -C _1-6 alkyl,
2) -C _2-6 alkenyl,
3) -C _2-6 alkynyl,
4) -C _3-7 cycloalkyl,
5) -C _3-7 cycloalkenyl,
6) Heterocyclyl,
7) —C _1-6 alkyl-C _3-7 cycloalkyl
_{8) -C 1-6} alkyl - heterocyclyl,
9) Aryl, or
10) Heteroaryl
Where the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl and heteroaryl groups may each independently be substituted with one or two R ^A substituents. ;
R ¹ is
1) -H,
2) -C _1-6 alkyl,
3) -C _2-6 alkenyl,
4) -C _2-6 alkynyl,
5) -C _3-7 cycloalkyl,
6) -C _3-7 cycloalkenyl,
7) -C _1-5 perfluorinated product,
8) -heterocyclyl,
9) -aryl,
10) -heteroaryl,
11) -Benzyl, or
12) 5-[(3aS, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl] pentanoyl
Where alkyl, alkenyl, alkynyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each independently 1, 2 or 3 R ^A or R ¹ substituted Optionally substituted with a group;
R ² is
1) -H,
2) -C _1-6 alkyl,
3) ^-SR 1,
^{4) -C (O) R 1} ,
^{5) -S (O) R 1} ,
_{^{6) -S (O) 2 R}} 1,
7) -Benzyl (optionally substituted with ¹ , 2 or 3 R ^A or R ¹ substituents),
8) -L-heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both of L and the heteroaryl group),
9) -L-heterocyclyl ( which may be substituted with one or more R ^A or R ¹ substituents, and the substituent is bonded to one or both of L and heterocyclyl groups) ),
10) -L-aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both of the L and aryl groups),
11) -heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents), or
12) -Aryl (optionally substituted with one or more R ^A or R ¹ substituents), wherein each substituent is attached to the L group, if it is not already present. May be;
R ^A is
1) -halogen,
2) _-CF 3,
3) -OH,
4) ^-OR 1,
5) -L-OH,
6) ^-L-OR 1,
7) -OCF _3,
8) -SH,
9) ^-SR 1,
10) -CN,
11) _-NO 2,
12) _-NH 2,
13) -NHR ^1,
14) -NR ^{1 R 1,}
15) _-L-NH 2,
16) ^-L-NHR 1,
^{17) -L-NR 4 R 1} ,
18) ^-L-SR 1,
^{19) -L-S (O)} R 1,
_{20) -L-S (O)} 2 R 1,
21) -C (O) OH,
^{22) -C (O) OR 1} ,
_{23) -C (O) NH 2} ,
^{24) -C (O) NHR 1} ,
25) —C (O) N (R ¹ ) R ¹ ,
^{26) -NHC (O) R 1} ,
^{27) -NR 1 C (O)} R 1,
^{28) -NHC (O) OR 1} ,
^{29) -NR 1 C (O)} OR 1,
_{30) -OC (O) NH 2} ,
^{31) -OC (O) NHR 1} ,
32) —OC (O) N (R ¹ ) R ¹ ,
^{33) -OC (O) R 1} ,
^{34) -C (O) R 1} ,
_{35) -NHC (O) NH 2} ,
36) -NHC (O) NHR ¹ ,
37) -NHC (O) N (R ¹ ) R ¹ ,
^{38) -NR 1 C (O)} NH 2,
^{39) -NR 1 C (O)} NHR 1,
^{40) -NR 1 C (O)} N (R 1) R 1,
_{^{41) -NHS (O) 2 R}} 1,
_{^{42) -NR 1 S (O)}} 2 R 1,
_{43) -S (O) 2 NH} 2,
44) -S (O) ₂ NHR ¹ ,
_{^{45) -S (O) 2 N}} (R 1) R 1,
^{46) -S (O) R 1} ,
_{^{47) -S (O) 2 R}} 1,
_{^{48) -OS (O) 2 R}} 1,
_{^{49) -S (O) 2 OR}} 1,
50) -benzyl,
51) -N _3, or
52) -C (-N = N - ) (CF 3)
Wherein the benzyl group may be substituted with ¹ , 2 or 3 R ^A or R ¹ substituents, or a salt or prodrug thereof.
Aspect 2 General Formula III or IV:
Wherein Z and R ² are each as defined in Embodiment 1, m is an integer from 1 to 6, and when m is 2 or more, X _i is the same or different and each independently NR ¹ , CH ₂ , O, or S, wherein R ¹ is as defined in embodiment 1, _Li is the same or different and each independently is L as defined in embodiment 1,
And R ³ and R ⁴ are the same or different and are each independently H, R ¹ as defined in embodiment 1 , or together with N, one selected from N, O and S Or a 3- to 7-membered ring which may contain a plurality of other heteroatoms, and the ring may be substituted with one or a plurality of R ¹ or R ^A ], or Its salt or prodrug.
Aspect 3 General Formula V or VI:
Or a salt or prodrug thereof , wherein Z, L, R ¹ and R ² are as defined in Aspect 1;
Embodiment 4 Formula IIA:
[Wherein R ¹ , W and R ² are as defined in Aspect 1], or a salt or prodrug thereof.
Embodiment 5 Formula IIB:
[Wherein W and R ² are each as defined in Aspect 1, and Het may be substituted with ^one or more R ¹ or R ^A as defined in Aspect 3 A 7-membered heterocycle], or a salt or prodrug thereof.
Embodiment 6 Formula IIC:
[Wherein W and R ² are each as defined in Aspect 1;
And R ⁵ and R ⁶ are the same or different and are each independently L as defined in the embodiment 1, or together with C, one or more selected from N, O and S Or a salt or prodrug thereof, which forms a 5- to 7-membered ring optionally containing a heteroatom of which is optionally substituted with ^one or more R ¹ or R ^A .
Embodiment 7 The compound according to embodiment 6, wherein the ring is a 5-membered ring and the heteroatom is N.
Aspect 8. A compound according to aspect 6 or 7, wherein the ring comprises 4 Ns.
Aspect 9 The compound according to any one of aspects 6 to 8, wherein R ² is benzyl.
Embodiment 10 Formula IVA:
[Wherein W, L, R ¹ and R ² are as defined in Aspect 1;
And m, L _i , R ³ and R ⁴ are as defined in Aspect 2, respectively, or a salt or prodrug thereof.
Embodiment 11 Formula VIA:
[Wherein Z, L, R ¹ and R ² are as defined in Aspect 1;
And R ³ and R ⁴ are as defined in Aspect 2, respectively, or a salt or prodrug thereof.
Aspect 12 Z is, be _CO 2 Me or 2-methyl -2H- tetrazol-5-yl;
R ² is benzyl, 3-thienylmethyl or 3-pyridinylmethyl; and
W is NH-L-N (R ¹ ) R ¹ , wherein L is C _2-4 alkyl and R ¹ is C _1-4 alkyl or (R ¹ ) and R ¹ Together with the nitrogen atom to which they are attached form a 3-7 membered ring that may contain one or more other heteroatoms selected from N, O and S; The compound according to embodiment 1, wherein the ring is optionally substituted with ^one or more R ¹ or R ^A.
Aspect 13
Or a salt or prodrug thereof.
Aspect 14
Or a salt or prodrug thereof.
Aspect 15 A pharmaceutical composition comprising the compound defined in any one of aspects 1 to 14, or a salt or prodrug thereof, and a pharmaceutically acceptable carrier.
Embodiment 16 Use of the compound defined in any one of Embodiments 1 to 14 for hematopoietic stem cell proliferation.
Embodiment 17 Use according to embodiment 16, wherein the hematopoietic stem cells are human cells.
Embodiment 18 A method of increasing stem cells and progenitor cells, comprising culturing a starting population comprising hematopoietic stem cells (HSC) with an agent capable of increasing the number of HSCs, the agent comprising: A method comprising a compound as defined in any one of the paragraphs.
Embodiment 19 The method according to embodiment 18, in vivo, in vitro or ex vivo.
Embodiment 20 The method according to embodiment 18 or 19, wherein the starting cell population comprises CD34 + cells taken from mobilized peripheral blood (mPB), bone marrow (BM) or umbilical cord blood (UCB).
Embodiment 21 A method of proliferating hematopoietic stem cells, wherein the method comprises converting a starting cell population into a biological molecule or another small molecule, optionally in the presence of at least one compound as defined in any one of embodiments 1-14. Culturing with at least one cell growth factor.
Embodiment 22 A cell population grown according to the method defined in any one of Embodiments 18 to 21.
Aspect 23. A pharmaceutical composition comprising a cell population grown using a compound as defined in any one of aspects 1-14.
Aspect 24 Any one of Aspects 1-14 for treating a hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immune deficiency disease in a subject in need of such treatment A method comprising administering a hematopoietic stem cell grown using a compound defined in the above, or a compound defined in any one of Embodiments 1 to 14 or a salt or prodrug thereof.
Aspect 25 A hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or inherited immunodeficiency disease is a bone marrow failure state, various congenital diseases of global interest (eg sickle cell anemia and thalassemia), lupus, Acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin's disease, aplastic anemia, 22. The method according to aspect 21, comprising true erythropoiesis, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiscott-Aldrich syndrome, inborn errors of metabolism (particularly Gaucher disease).
Aspect 26 Hematopoietic proliferated using a compound as defined in any one of aspects 1-14 for treating a hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immune deficiency disease in a subject Use of a stem cell, or a compound defined in any one of aspects 1 to 14, or a salt or prodrug thereof.
Aspect 27 A compound or a prodrug thereof as defined in any one of Aspects 1-14 in the manufacture of a medicament for treating a hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immune deficiency disease in a subject Use of.
Aspect 28. Use of a pharmaceutical composition as defined in aspect 15 for treating a hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immune deficiency disease in a subject.
Aspect 29 Hematopoietic disorders / hematopoietic malignancies, autoimmune diseases and / or hereditary immunodeficiency diseases are bone marrow failure states, various congenital diseases of global interest (eg sickle cell anemia and thalassemias), lupus, Acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin's disease, aplastic anemia, 29. Any one of aspects 26-28, including true erythropoiesis, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiscott-Aldrich syndrome, inborn errors of metabolism (especially Gaucher disease). use.
Aspect 30 Hematopoietic proliferated using a compound as defined in any one of Aspects 1-14 for treating a hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immune deficiency disease in a subject A stem cell, or a compound defined in any one of aspects 1 to 14, or a salt or prodrug thereof.
Aspect 31 Hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immunodeficiency disease is a bone marrow failure condition, various congenital diseases of global interest (eg sickle cell anemia and thalassemia), lupus, Acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin's disease, aplastic anemia, A hematopoietic stem cell or compound as defined in embodiment 30 comprising true erythropoiesis, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiscott-Aldrich syndrome, inborn errors of metabolism (especially Gaucher disease).
Embodiment 32 The pharmaceutical composition according to embodiment 15 or 23, which is suitable for intravenous infusion.
Aspect 33 A kit for use in increasing stem cells and progenitor cells, comprising the compound defined in any one of aspects 1 to 14 and instructions for use.
Aspect 34 A kit for use in stem cell expansion comprising a compound as defined in any one of aspects 1-14 and instructions for use, wherein the kit is at least a biomolecule or another small molecule A kit that may contain one cell growth factor.

Claims (27)

General formula:
[Where:
Z is
^{2) -C (O) OR 1} ,
^{3) -C (O) NHR 1} ,
4) —C (O) N (R ¹ ) R ¹ ,
6) -CN, or 18) -heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents),
Here, when (R ¹ ) and R ¹ are bonded to a nitrogen atom, they together with the nitrogen atom contain one or more other heteroatoms selected from N, O and S May form a 3-7 membered ring, which ring may be substituted with ^one or more R ¹ or R ^A ;
W is
3) ^-OR 1,
9) -NHR ¹ ,
^{10) -N (R 1) R} 1,
13) -L-N (R ¹ ) R ¹ ,
48) -L-heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both of the L and heterocyclyl groups) ,
58) -OL-heterocyclyl (which may be substituted with one or more R ^A or R ¹ substituents, wherein the substituent is bonded to either or both of the L and heterocyclyl groups; Is)
_{^{74) - (N (R 1}} ) -L) n -N (R 1) R 1, where, n is a 1,
78)-(N (R ¹ ) -L) _n -heterocyclyl (optionally substituted with one or more R ^A or R ¹ substituents), where n is 1.
Where each substituent may be attached to the L group, if it is not already present,
And when two R ¹ substituents are present on the same nitrogen atom, each R ¹ substituent is independently selected from the list of R ¹ values described below,
When the (R ¹⁾ and R ¹ is bonded to the nitrogen atom, they, together with the nitrogen atom, may form a 3- to 7-membered ring, which ring may one or more Optionally substituted with R ¹ or R ^A of
L is
1) -C _1-6 alkyl, wherein alkyl is ^optionally substituted with one or two R ^A substituents,
4) -C _3-7 cycloalkyl,
6) Heterocyclyl,
Where the alkyl, cycloalkyl, and heterocyclyl groups may each independently be substituted with one or two R ^A substituents;
R ¹ is
1) -H,
2) -C _1-6 alkyl,
4) -C _2-6 alkynyl,
7) -C _1-5 perfluorinated alkyl,
8) -heterocyclyl,
9) -aryl, or 12) 5-[(3aS, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl] pentanoyl, wherein alkyl, Each perfluorinated alkyl, heterocyclyl, aryl group may be independently substituted with ¹ , 2, or 3 R ^A or R ¹ substituents;
R ² is
1) -H,
2) -C _1-6 alkyl,
^{4) -C (O) R 1} ,
7) -Benzyl (optionally substituted with ¹ , 2 or 3 R ^A or R ¹ substituents),
8) -L-heteroaryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both of L and the heteroaryl group),
10) -L-aryl (optionally substituted with one or more R ^A or R ¹ substituents, the substituents being bound to either or both of the L and aryl groups),
Where each substituent may be attached to the L group if it is not already present;
R ^A is
1) -halogen,
2) _-CF 3,
3) -OH,
4) ^-OR 1,
12) _-NH 2,
13) -NHR ^1,
14) -NR ^{1 R 1,}
15) _-L-NH 2,
16) ^-L-NHR 1,
^{34) -C (O) R 1} ,
Or 52) -C (-N = N - ) (CF 3)
Where heteroaryl is a 3-10 membered monocyclic or bicyclic system having at least one aromatic ring and 1-4 heteroatoms selected from N, S and O; The heterocycle is a 3 to 7-membered non-aromatic ring containing 1 to 4 heteroatoms selected from N, S and O], or a salt thereof, except for the following compounds.
Formula IIA, Formula IIB or Formula IIC:
Wherein R ¹ , W and R ² are each as defined in claim 1 and Het is substituted with ^one or more R ¹ or ^RA as defined in claim 1. An optionally 3 to 7 membered heterocycle, wherein R ⁵ and R ⁶ are the same or different and are each independently L as defined in claim 1 or together with C, N A 5- to 7-membered ring optionally containing one or more heteroatoms selected from O and S, which ring is substituted with ^one or more R ¹ or R ^A The compound according to claim 1, which is a compound of or a salt thereof. The compound according to claim 2, wherein the ring formed by combining R ⁵ and R ⁶ is a 5-membered ring containing 4 N atoms. R ² is benzyl, a compound according to any one of claims 1 to 3. The compound of claim 1, wherein R ² is H. W is the following formula:
The compound of claim 1, wherein Formula VIA:
Wherein Z, L, R ¹ and R ² are each as defined in claim 1;
R ³ and R ⁴ are the same or different and are independently H, R ¹
Or R ³ and R ⁴ together with N form a ^{3- to} 7-membered ring which may contain one or more heteroatoms selected from N, O and S, and the ring May be substituted with ^one or more R ¹ or R ^A ], or a salt thereof. Z is a _CO 2 Me or 2-methyl -2H- tetrazol-5-yl;
R ² is benzyl, 3-thienylmethyl or 3-pyridinylmethyl; and W is NH—LN (R ¹ ) R ¹ , wherein L is C _2-4 alkyl, R ¹ Is C _1-4 alkyl, or (R ¹ ) and R ¹ together with the nitrogen atom to which they are attached, one or more other selected from N, O and S The compound according to claim 1, which forms a 3- to 7-membered ring which may contain a hetero atom, and the ring may be substituted with ^one or more R ¹ or R ^A. Or a salt thereof. The compound has the following formula:
Or a pharmaceutically acceptable salt thereof. The compound has the following formula:
The compound of claim 1, which is a hydrobromide salt of The pharmaceutical composition for hematopoietic stem cell proliferation containing the compound defined in any one of Claims 1-11, or its salt. The composition according to claim 12, wherein the hematopoietic stem cells are human cells. A method of proliferating stem cells and progenitor cells, wherein the method comprises starting cell populations, optionally with biomolecules or agents, together with an agent comprising a compound as defined in any one of claims 1 to 11, or a salt thereof. Culturing in vitro or ex vivo with at least one cell growth factor which is a small molecule of 15. The method of claim 14, wherein the starting cell population comprises CD34 + cells taken from mobilized peripheral blood (mPB), bone marrow (BM) or umbilical cord blood (UCB). 16. The method of claim 14 or 15, wherein the starting cell population consists essentially of CD34 + cells purified from one or two umbilical cord blood units. The method according to any one of claims 14 to 16, wherein the starting cell population is cultured for 2 to 21 days in the presence of the compound. 18. A method according to any one of claims 14 to 17, wherein the compound is administered to the starting cell population at a concentration between 1 nM and 3000 nM. The biomolecules are interleukin-3 (IL-3), granulocyte / macrophage colony stimulating factor (GM-CSF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), stem cell factor (SCF) 19. The method of any one of claims 14-18, comprising, interleukin-6 (IL-6), or a combination thereof. 20. The method of claim 19, wherein the biomolecule comprises SCF, FLT3-L, TPO, IL-6 or a combination thereof . 15. The method of claim 14, wherein the other small molecule is StemRegenin 1. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 11, or a salt thereof, and a pharmaceutically acceptable carrier. A pharmaceutical composition for treating a hematopoietic disorder / hematopoietic malignant disease, autoimmune disease and / or hereditary immunodeficiency disease in a subject, the compound defined in any one of claims 1 to 11, or a compound thereof A pharmaceutical composition comprising a salt. Hematopoietic disorders / hematopoietic malignancies, autoimmune diseases and / or hereditary immunodeficiency diseases are bone marrow failure states, various congenital diseases of global interest, lupus, acute myeloid leukemia, acute lymphoblastic leukemia Chronic myelogenous leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin's disease, aplastic anemia, true erythropoiesis, hemoglobinuria, fanconi 24. The pharmaceutical composition according to claim 23 , comprising anemia, thalassemia, sickle cell anemia, Wiscot-Aldrich syndrome, inborn error of metabolism . 25. The pharmaceutical composition according to claim 24, wherein the hematopoietic disorder / hematopoietic malignancy, autoimmune disease and / or hereditary immune deficiency disease comprises sickle cell anemia or thalassemia. 25. The pharmaceutical composition according to claim 24, wherein the hematopoietic disorder / hematopoietic malignant disease, autoimmune disease and / or hereditary immune deficiency disease comprises Gaucher disease. A kit for use in the growth of hematopoietic stem cells, comprising a compound as defined in any one of claims 1 to 11, wherein the kit is at least one cell growth factor which is a biomolecule or another small molecule A kit that may contain.